Multiplexed detection with isotope-coded reporters

ABSTRACT

Some aspects of this invention provide reagents and methods for the sensitive, quantitative and simultaneous detection of target analytes in complex biological samples by liquid chromatography tandem mass spectrometry (LC MS/MS). Some aspects of this invention provide affinity reagents encoded with mass reporters for the sensitive and quantitative translation of an analyte of interest into a mass tag. The reagents and methods provided herein have general utility in analyte detection and encoding, for example, in biomolecular profiling, molecular diagnostics, and biochemical encoding.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/842,162, entitled “MULTIPLEXED DETECTION WITH ISOTOPE-CODEDREPORTERS” filed on Dec. 14, 2017, which is a continuation of U.S.application Ser. No. 14/005,416, entitled “MULTIPLEXED DETECTION WITHISOTOPE-CODED REPORTERS” filed on Jun. 12, 2014, which is a NationalPhase application under 35 U.S.C. § 371 of International Application No.PCT/US2012/029200, filed Mar. 15, 2012, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 61/452,908,entitled “MULTIPLEXED DETECTION OF ANALYTES WITH ISOTOPE-CODEDREPORTERS” filed on Mar. 15, 2011, which are herein incorporated byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant R01-CA124427awarded by the National Institutes of Health. The U.S. government hascertain rights in this invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(M065670233US03-SEQ-FL.xml; Size: 111,002 bytes; and Date of Creation:Dec. 6, 2022) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Mass spectrometry (MS) is an analytical technique that is useful fordetecting analytes in both a quantitative and a qualitative way. Duringthe MS process, detectable molecules, called mass tags, are ionized togenerate charged molecules or molecule fragments and subsequently themass-to-charge ratio of these molecules is measured.

MS is a highly versatile technology that can be used in variousscenarios of analyte detection, for example, in biomedical diagnostics,and environmental analysis. For example, MS can be used to determine thepresence or absence of an analyte, for example, a protein, nucleic acid,biomolecules, or small molecule compound, in a sample (for example, abiological sample), and/or to quantify an analyte in a sample.

While conventional MS assays are capable of detecting multiple analytes,e.g., peptides, simultaneously, quantitative analysis of multiplesamples, e.g. from different individuals or time points, is currentlylimited to 4-plex, 6-plex, and 8-plex formats. Further, differentanalytes often have widely varying physiochemical properties, and, thus,MS properties, which results in similarly wide variability insensitivity, specificity, and accuracy of MS based detection ofdifferent analytes. This variability in physiochemical properties ofdifferent analytes limits accurate simultaneous quantification of suchdifferent analytes in multiplex MS assays. The detection of multiplenaturally-occurring or endogenous analytes, the chemical structures, ofwhich often widely vary, also often necessitate monitoring a wide masswindow in a single MS assay, which can be very time-intensive. Further,the detection of target analytes in complex samples, such as biologicalsamples (e.g., blood, serum, or tissue biopsies), is often difficult andchallenging without extensive prior front-end processing prior to MSassays.

SUMMARY OF THE INVENTION

Some aspects of this invention address the shortcomings of conventionalMS technology by providing novel reagents and methods for thesimultaneous detection of a virtually unlimited number of analytes in asample, for example, a complex biological sample (e.g., a blood ortissue sample obtained from a subject) using MS methodology.

In some embodiments, this invention provides isotope coded reportermolecules, referred to herein as iCOREs, which are useful as mass tagsin multiplexed MS based analyte detection. In some embodiments, sets, orlibraries of iCOREs are provided that are useful in multiplexeddetection of a virtually unlimited number of analytes in a sample. Insome embodiments, the iCOREs in such a set or library are isobaric anddifferent iCOREs within such a set or library are distinguished by theirunique fragmentation ion signature. In some embodiments, differentfragmentation signatures are conferred to different iCOREs bydifferential isotope labeling. For example, in some embodiments, thisinvention provides a set or library of iCOREs, e.g., a set of isobaric,isotope-labeled, peptide mass tags, that are useful in the multiplexedMS based analyte detection methods provided herein. Methods for the useof iCOREs and sets or libraries of iCOREs are also provided herein.

Some aspects of this invention provide reagents and methods that areuseful for the translation of a parameter, for example, the presence ofa target analyte in a sample, the presence of a target activity in asample, or the identity of a sample, cell, or tissue, into an iCORE forMS detection, allowing for biochemical encoding of the parameter. Forexample, in some embodiments, this invention provides reagents andmethods for the simultaneous translation, or biochemical encoding, of aplurality of analytes, for example, analytes of varying a chemicalstructure, in a complex sample (e.g., a blood or tissue sample obtainedfrom a subject) into a plurality of iCOREs, for example, a plurality ofisobaric, isotope-labeled, peptide mass tags, so that each analyte isrepresented by a different iCORE having a characteristic fragmentationsignature. Accordingly, each analyte can be identified by detecting thefragmentation signature associated with the specific iCORE encoding theanalyte, for example, in an MS/MS assay. To give another example, insome embodiments, this invention provides reagents and methods for thesimultaneous translation, or biochemical encoding, of a plurality ofbiological activities, for example, enzymatic activities (e.g.,protease, kinase, or phosphatase activities), in a complex sample (e.g.,a blood or tissue sample obtained from a subject) into a plurality ofiCOREs, for example, a plurality of isobaric, isotope-labeled, peptidemass tags, so that each activity is represented by a different iCOREhaving a characteristic fragmentation signature. Accordingly, eachactivity can be identified by detecting the fragmentation signatureassociated with the specific iCORE encoding the activity, for example,in an MS/MS assay.

Each of the embodiments of the invention can encompass variousrecitations made herein. It is, therefore, anticipated that each of therecitations of the invention involving any one element or combinationsof elements can, optionally, be included in each aspect of theinvention. Similarly, each aspect or embodiment of the invention can beexcluded from any other aspect or embodiment, or any combination ofaspects or embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Schematic of an exemplary method for multiplexedtranslational analyte detection. The displayed method includes anoptional enrichment step.

FIG. 2 . Utilization of a canonical peak from glu-fib MS/MS spectra asreporter. The fragmentation spectrum of glu-fib and an exemplaryencoding strategy is shown. EGVNDNEEGFFSAR corresponds to SEQ ID NO: 1.

FIG. 3 . MS and MS/MS data obtained from a 10-plex iCORE libraryexperiment. EGVNDNEEGFFSAR corresponds to SEQ ID NO: 1.

FIG. 4 . Multiplex, quantitative detection of iCOREs in library.

FIG. 5 . Evaluation of the dynamic range of multiplexed iCORE assays.

FIG. 6 . Photocleavage and photocleavage efficiency. EGVNDNEEGFFSARcorresponds to SEQ ID NO: 1. KGGPVGLIGC corresponds to SEQ ID NO: 2.

FIG. 7 . Exemplary chemistry for the generation of PEG hydrogel-embeddediCORE tags.

FIGS. 8A-8C. Schematic of iCORE approach for urinary diagnostics.

FIGS. 9A-9C. Long-circulating iron oxide nanoworm chaperones.

FIGS. 10A-10E. Selecting protease-sensitive substrates for NW-chaperonedurinary trafficking.

FIGS. 11A-11H. Urinary biomarkers of hepatic fibrosis and resolution inDDC-treated mice.

FIGS. 12A-12D. Immunofluorescence of liver sections.

FIGS. 13A-13F. Photo-caged iCORE libraries for multiplexed profiling ofprotease activities by LC MS/MS. EGVNDNEEGFFSAR corresponds to SEQ IDNO: 1. KGGPWGIWGQGGC corresponds to SEQ ID NO: 3.

FIGS. 14A-14B. Isobaric COded REporter (iCORE) mass encoding.EGVNDNEEGFFSAR corresponds to SEQ ID NO: 1.

FIG. 15 . MS/MS spectrum of 10-plex iCORE library. iCORE peak clusterscentered on y-type ions. The y6 region outlined by a box is presented asFIG. 13 e.

FIGS. 16A-16B. Unit collection window for peptide fragmentationminimizes peak overlap arising from naturally occurring isotopes. (a) Atypical MS spectrum of an isotope-coded Glu-fib peptide. The parentprecursor peptide was collected for fragmentation via a unit mass window(gray), excluding the naturally occurring isotope peaks. (b) ResultingMS/MS spectrum. Isotope peak was minimized, comprising ˜5% of theoriginal peak intensity. EGVNDNEEGFFSAR corresponds to SEQ ID NO: 1.GFFSAR corresponds to SEQ ID NO: 4.

FIGS. 17A-17C. iCORE LC MS/MS analysis is quantitative.

FIGS. 18A-18B. Protease-specific iCORE mass signatures. (a) iCORE MS/MSprofiles of recombinant proteases MMP2, MMP12, and thrombin. (b)Graphical representation of Pearson's correlation coefficients betweenproteases.

FIG. 19 . iCORE profiles of control animals. Box-and-whisker plots ofindividual iCORE peak intensities (repeated measures ANOVA, n=5)

FIGS. 20A-20B. ROC curves of fibrosing biomarkers.

FIGS. 21A-21B. ROC curves of resolving biomarkers.

FIG. 22 . In vitro CEA secretion by LS 174T human colorectal cancercells. Quantification of CEA from media at days 1 and 2 by ELISA.

FIGS. 23A-23G. Synthetic biomarkers outperform serum CEA for earlycancer detection.

FIGS. 24A-24B. NW accumulation in tumor tissue. (a) VivoTag-680-labeledNWs or saline solutions were injected into LS 174T xenograft animals.Following excision, the tumors were scanned for NW accumulation. (b)Immunofluorescence analysis of tumor sections for blood vessels and NW.Scale bar=50 μm.

FIGS. 25A-25B. ROC curves of tumor biomarkers.

DEFINITIONS

The term activity, as used herein, refers to a biological activity. Theterm includes, in some embodiments, enzyme activity, for example,hydrolase, transferase, lyase, isomerase, ligase, or oxidoreductaseactivity. In some embodiments, the activity is a protease activity. Insome embodiments, the activity is a phosphatase activity. In someembodiments, the activity is a kinase activity. Enzymatic activity canbe encoded in iCOREs, for example, by providing a substrate of thetarget enzyme comprised in an iCORE, exposing the iCORE to a samplecomprising the enzyme, and detecting the enzyme-modified substrate, forexample, by methods described herein. Typically, an iCORE for encodingan enzyme activity comprises a target substrate of that enzyme (e.g., aprotease recognition site, or a phosphorylation site, etc.), and themodification of the target site by the enzyme (e.g., cleavage,phosphorylation or de-phosphorylation) can be detected, for example, bymethods described herein.

The term analyte, as used herein, refers to a molecule the presence orabsence or the quantity of which is subject to analysis. Typically, ananalyte is a molecule of interest, for example, a protein or peptide, anucleic acid molecule, a carbohydrate, a lipid, a metabolite, a smallorganic molecule, a drug, or a drug derivative (e.g. a drug metabolite),a cell surface marker, or a secreted molecule, the detection orquantification of which is of interest to a researcher or clinician, forexample, for research or diagnostic purposes. A target analyte is ananalyte the presence, absence, or the quantity of which in a sample, forexample, in a biological, experimental, or environmental sample issubject to analysis. An analyte may be a biomarker, for example, abiomarker the presence, absence, or quantity of which in a sampleindicates a particular condition of the sample or the subject,experiment, or environment the sample was obtained from. In someembodiments, an analyte is a biomedical biomarker, for example, aprotein, peptide, polysaccharide, small molecule, or metabolite in asample obtained from a subject diagnosed with or suspected to have adisease or condition, wherein the presence, absence, or quantity of thebiomarker in the sample is indicative of the presence, absence, or stateof the disease or condition in the subject. For example, in someembodiments, a diagnostic assay provided herein comprises the detectionof a panel of protein and metabolite biomarkers the presence, absence,or quantity of which in a blood or serum sample obtained from a subjectis indicative of the presence, absence, or state of a cancer, or aplurality of cancers in the subject. The protein and metabolitebiomarkers investigated in such an assay would be the target analytes ofthat assay.

The term antibody, as used herein, refers to an immunoglobulin, whethernatural or wholly or partially synthetically produced. Antibodyderivatives which maintain specific binding ability, for example,antigen-binding antibody fragments, such as Fab, Fab′, or F(ab′)2fragments, or engineered antibodies, such as scFvs, are also includedreferred to by the term antibody. The term also refers to any proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin binding domain. These proteins may be derived fromnatural sources, or partly or wholly synthetically produced. An antibodymay be monoclonal or polyclonal. An antibody may be a member of anyimmunoglobulin class, including, but not limited to, any of the humanclasses: IgG, IgM, IgA, IgD, and IgE.

The term antibody fragment, as used herein, refers to any derivative ofan antibody which is less than full-length. Preferably, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv,diabody, single variable domain, and Fd fragments. The antibody fragmentmay be produced by any means. For instance, the antibody fragment may beenzymatically or chemically produced by fragmentation of an intactantibody or it may be recombinantly produced from a gene encoding thepartial antibody sequence. Alternatively, the antibody fragment may bewholly or partially synthetically produced. The antibody fragment mayoptionally be a single chain antibody fragment. Alternatively, thefragment may comprise multiple chains which are linked together, forinstance, by disulfide linkages. The fragment may also optionally be amulti-molecular complex. A functional antibody fragment will typicallycomprise at least about 50 amino acids and more typically will compriseat least about 200 amino acids. Single-chain Fvs (scFvs) are recombinantantibody fragments consisting of only the variable light chain (VL) andvariable heavy chain (VH) covalently connected to one another by apolypeptide linker. Either VL or VH may be the NH2-terminal domain. Thepolypeptide linker may be of variable length and composition so long asthe two variable domains are bridged without serious stericinterference. Typically, the linkers are comprised primarily ofstretches of glycine and serine residues with some glutamic acid orlysine residues interspersed for solubility. Diabodies are dimericscFvs. The components of diabodies typically have shorter peptidelinkers than most scFvs, and they show a preference for associating asdimers. An Fv fragment is an antibody fragment which consists of one VHand one VL domain held together by non-covalent interactions. The termdsFv is used herein to refer to an Fv with an engineered intermoleculardisulfide bond to stabilize the VH-VL pair. An F(ab′)2 fragment is anantibody fragment essentially equivalent to that obtained fromimmunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH4.0-4.5. The fragment may be recombinantly produced. A Fab′ fragment isan antibody fragment essentially equivalent to that obtained byreduction of the disulfide bridge or bridges joining the two heavy chainpieces in the F(ab′)2 fragment. The Fab′ fragment may be recombinantlyproduced. A Fab fragment is an antibody fragment essentially equivalentto that obtained by digestion of immunoglobulins (typically IgG) withthe enzyme papain. The heavy chain segment of the Fab fragment is the Fdpiece.

The term binding agent, as used herein, refers to a molecule that bindsto another molecule with high affinity. In some embodiments, the bindingis through non-covalent interaction. In some embodiments, the binding isspecific, meaning that the binding agent binds only one particular typeof molecule, or a narrow class of highly similar molecules with highaffinity. Non-limiting examples of binding agents are antibodies,antibody fragments, aptamers, and adnectins.

The term body fluid, as used herein, refers to any body fluid including,without limitation, serum, plasma, lymph fluid, synovial fluid,follicular fluid, seminal fluid, amniotic fluid, mild, whole blood,sweat, urine, cerebrospinal fluid, saliva, semen, sputum, tears,perspiration, mucus, tissue culture medium, tissue extracts, andcellular extracts. It may also apply to fractions and dilutions of bodyfluids. The source of a body fluid can be a human body, an animal body,an experimental animal, a plant, or other organism.

The term conjugated, as used herein, refers to a state of relativelystable association between two entities, for example, between an iCOREand a binding agent. In some embodiments, conjugated entities are linkedby a direct or indirect covalent or non-covalent interaction.Preferably, the association is via covalent bond. In some embodiments,two peptides are conjugated via protein fusion, e.g., a peptide iCORE asprovided herein, may be conjugated to a peptidic binding agent, e.g., anantibody or antibody fragment, by fusing the iCORE to the binding agent,e.g., by expression of a recombinant iCORE-binding agent fusion protein.Non-covalent interactions that result in conjugation include, but arenot limited to hydrogen bonding, van der Waals interactions, hydrophobicinteractions, magnetic interactions, and electrostatic interactions.Typically, two conjugated entities are associated with each other in amanner stable enough to withstand the conditions typically encounteredduring an iCORE experiment, for example, an iCORE conjugated to abinding agent is associated with the binding agent in a mannersufficient for the bond between the two to endure the binding andwashing steps typically comprised in the methods they are used in, forexample, before the two entities are intentionally separated (e.g., bycleaving a linker connecting the iCORE to the binding agent).

The term enriched, as used herein, refers to a sample or composition inwhich the proportion of a material of interest in a mixture of materialscomprising both the material of interest and at least one additionalmaterial is increased as compared to the original proportions in thesample. Such an increase can be achieved by methods of physicalseparation, chemical interaction or reaction, and other methods wellknown to those of skill in the art, or provided herein. For example, anoriginal sample comprising a population of different iCOREs conjugatedto different binding agents may be enriched to include predominantlythose iCOREs conjugated to binding agents that bind to their specifictarget analytes by immobilizing the analyte-bound iCOREs on a solidsupport and washing away all or most of the unbound iCOREs. Isolation orpurification are within the scope of the term, but such steps are notrequired in order to enrich a compound, e.g., a desired iCORE.

The term fragmentation signature, or fragmentation ion signature, asused herein, refers to the pattern of ions that an iCORE can fragmentinto, for example, during an MS assay, e.g., an MS/MS assay. In someembodiments, differential isotope labeling of iCOREs of the same basesequence, e.g., of the same amino acid sequence in the case of peptideiCOREs, is used to produce a set of different isobaric iCOREs, each ofwhich produces at least one ion, for example, a y₇ ion, that can bedistinguished from other ions of the same type, e.g., y₇ ions, producedby the other iCOREs of the set, e.g., in an MS assay, e.g. an MS/MSassay. For example, the ten exemplary iCOREs G1-G10 described in FIG. 3each exhibit a unique fragmentation signature, each producing adifferent mass peak representing the y₇ fragment of the iCOREs in anMS/MS assay. A unique fragmentation signature is a signature of a givenpolymeric molecule that results in a unique fragmentation ion, forexample, a unique y₇ fragmentation ion, which can be unambiguouslyidentified, or which can be distinguished from any other fragmentationion, e.g., any other y₇ ion produced by a library of iCOREs. In someembodiments, different fragmentation signatures are conferred todifferent iCOREs by differential isotope labeling. For example, in someembodiments, an isobaric iCORE library is produced by distributing heavyisotopes across the iCORE molecule, e.g., across the amino acid residuesof a peptide iCORE, in a way that each different iCORE produces areporter fragmentation ion (e.g., a y₇ ion) of a different mass, whilethe whole sequence, (e.g., the peptide sequence comprising the reporterand the balance) of all iCOREs is of the same mass.

The term iCORE, as used herein, refers to an isotope-coded reportermolecule that can be used as a mass tag in an MS assay, for example, anMS/MS assay. Typically, an iCORE is an isotope-labeled polymer, forexample, a polypeptide, polynucleotide, or polysaccharide that comprisesat least 5 monomeric residues, e.g. amino acid, nucleotide, ormonosaccharide residues. In some embodiments, an iCORE comprises morethan 5 monomeric residues. In some preferred embodiments, the structureof an iCORE allows for the generation of different fragmentationsignatures, for example, by differential isotope labeling of a monomericresidue, or a combination of monomeric residues. In some embodiments,the polymer structure, e.g., the amino acid, nucleotide, ormonosaccharide sequence of an iCORE allows for the generation of atleast about 10 iCORE molecules, for example, isobaric iCORE molecules,having different fragmentation signatures that can be distinguished inan MS assay, e.g., an MS/MS assay. In some embodiments, the polymerstructure allows for the generation of more than 10 different iCOREshaving different fragmentation signatures.

The term isobaric, as used herein, refers to a group of molecules havingthe same molecular weight. For example, in some embodiments, a set ofisobaric peptide iCOREs may be a set of peptides that all have the sameweight, but different fragmentation signatures.

The term parameter, as used herein in the context of iCORE encoding,refers to a characteristic, feature, or measurable factor in a sample,for example, in a biological sample. In some embodiments, the termincludes the presence, absence, or quantity of an analyte. In someembodiments, the term includes the presence, absence or quantity of abiological activity, for example, an enzymatic activity or a bindingactivity.

The term plurality, as used herein, refers to two or more of theelements so qualified.

The term polynucleotide, which is used herein interchangeably with theterms oligonucleotide and nucleic acid molecule herein, refers to apolymer of nucleotides. The polymer may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine,4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine,methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologicallymodified bases (e.g., methylated bases), intercalated bases, modifiedsugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,2′-O-methylcytidine, arabinose, and hexose), or modified phosphategroups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term polypeptide is used herein interchangeably with the termspeptide, oligopeptide, and protein, and refers to a polymer of aminoacid residues linked together by peptide bonds. The term, as usedherein, refers to proteins, polypeptides, and peptides of any size,structure, or function. Typically, a polypeptide is at least three aminoacids long. In some embodiments, polypeptides, for example, peptideiCOREs, comprise naturally-occurring amino acids, althoughnon-naturally-occurring amino acids (e.g., compounds that do not occurin nature but that can be incorporated into a polypeptide chain and/oramino acid analogs as are known in the art may alternatively beemployed. Also, one or more of the amino acids in an inventivepolypeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a hydroxyl group, a phosphategroup, a farnesyl group, an isofarnesyl group, a fatty acid group, alinker for conjugation, functionalization, or other modification, etc. Apeptide may be isotope-labeled. A peptide may comprise D-amino acids,L-amino acids, or a mixture of D-amino acids and L-amino acids. In someembodiments, a D-amino acid or a plurality of D-amino acids in apolypeptide confers increased protease resistance to the respectivepolypeptide as compared to a polypeptide of the same sequence butconsisting of L-amino acids. A polypeptide may also be a single moleculeor may be a multi-molecular complex. A polypeptide may be a fragment ofa naturally occurring protein or peptide. A polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination of these.

The term polysaccharide, as used herein, refers to a polymer of sugars.Typically, a polysaccharide comprises at least two sugars. The polymermay include natural sugars (e.g., glucose, fructose, galactose, mannose,arabinose, ribose, and xylose) and/or modified sugars (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose).

The term sample, as used herein, refers to a composition of matterrepresentative of a biological, clinical, or experimental environment.For example, a biological sample may be a sample obtained from asubject, such as a body fluid sample, or a cell or tissue sample, or toa sample obtained from an experimental environment, such as acomposition comprising a small molecule compound, a cell culturesupernatant, a composition comprising an engineered organ, and so forth.A complex sample is a sample comprising a plurality of differentanalytes and/or of non-analyte molecules in addition to an analyte.Non-limiting examples of complex samples are a serum sample, a bloodsample, a urine sample, and a tissue sample. In some embodiments, acomplex sample is a sample comprising such a large number of molecules(e.g. analytes or non-analyte molecules) that the detection of a singletarget analyte, for example, a peptide or metabolite, cannot readily beachieved by an MS/MS assay, for example, because of interference ofother molecules with similar MS signatures. In some embodiments, acomplex sample may require extensive front-end processing, for example,target analyte enrichment, to allow detection of a target analyte in aconventional MS assay.

The term small molecule, which is used herein interchangeably with theterms small molecule compound, and drug, refers to a compound eithersynthesized in the laboratory or found in nature, which is typicallycharacterized in that it contains several carbon-carbon bonds, and has amolecular weight of less than 1500, although this characterization isnot intended to be limiting for the purposes of the present invention.Examples of small molecules that occur in nature include, but are notlimited to, taxol, dynemicin, and rapamycin. Examples of small moleculesthat are synthesized in the laboratory include, but are not limited to,compounds described in Tan et al., (“Stereoselective Synthesis of overTwo Million Compounds Having Structural Features Both Reminiscent ofNatural Products and Compatible with Miniaturized Cell-Based Assays” J.Am. Chem. Soc. 1998, 120, 8565) and U.S. Pat. No. 7,109,377, entitled“Synthesis of Combinatorial Libraries of Compounds Reminiscent ofNatural Products”, the entire contents of which are incorporated hereinby reference. In certain other preferred embodiments,natural-product-like small molecules are utilized.

The term subject, as used herein, refers to a human, a non-humanprimate, a non-human mammal (e.g., a cow, a horse, a pig, a sheep, agoat, a dog, a cat, or a rodent), a vertebrate, an arthropod, achordate, an annelid, a mollusk, a nematode, an echinoderm. In someembodiments, a subject is a laboratory animal, e.g., a mouse, rat, cat,dog, pig, cow, hamster, gerbil, frog, fish, worm (e.g., C. elegans), orfly (e.g., fruit fly, D. melanogaster). In some embodiments, a subjectis a microorganism, for example, a yeast, bacteria, or fungus. In someembodiments, for example, in some embodiments involving a clinicalapplication of an aspect of this invention, the subject is diagnosedwith or suspected to have a disease or condition.

DETAILED DESCRIPTION OF THE INVENTION

Some aspects of this invention provide methods and reagents for encodinga plurality of biological parameters, for example, the presence orabsence of an analyte, or of an enzymatic activity in a sample, intomass-encoded reporters, termed iCOREs. This allows for, e.g., themultiplexed detection of analytes or enzymatic activities in complexbiological samples. The exogenous reporters can be analyzed in a singleassay, e.g., an MS/MS assay, thus avoiding the need for multiple assaysof different types, as often required when assessing different analytesor enzymatic activities in biological samples using endogenousreporters.

The dependence on endogenous reporters to indicate disease in biologicalsamples is a major limitation to diagnostic approaches. Herein, thedevelopment of iCOREs, exogenous mass-encoded reporters, as ‘syntheticbiomarkers’ of disease is described. In some embodiments, iCOREsdescribed herein are used for in vivo diagnostics. In some embodiments,iCOREs are designed to perform three functions in vivo (uponadministration to a subject): target sites of disease, sampledysregulated protease activities, and emit mass-encoded reporters intohost urine for multiplexed detection by mass spectrometry. Todemonstrate exemplary applications of this technology, it was applied toa xenobiotic model of liver fibrosis as a noninvasive alternative tobiopsy-based monitoring, and sensitive and specific synthetic biomarkerswere identified that report on both actively fibrosing and resolvingstages of liver disease. Different iCORE panels were also identifiedthat markedly lowered the threshold for early cancer detection whencompared with blood biomarkers in a mouse model of colorectal cancer.The ability to rapidly design, screen and identify iCOREs as syntheticbiomarkers for precise, multiplexed monitoring of disease apart fromendogenous biomarkers is broadly amenable to distinct pathophysiologicalprocesses, with additional applications in systems biology, drugdevelopment and point-of-care diagnostics.

Biomarker discovery is motivated by the desire to identify reliableindicators of disease for risk assessment, early detection, predictingpatient responses to therapies, and surveillance of recurrentdisease.^(1,2) To date, a broad range of distinct biological speciessuch as metabolites,³ peptides,⁴ proteins,^(2,5) cell-free nucleicacids,⁶ exosomes,⁷ and circulating tumor cells,⁸ have been developedinto biomarkers with varying degrees of performance. However, thereliance on native components to indicate disease is limited byfundamental technical and biological challenges because biomarkers arefrequently found in low levels in circulation,^(8,9) are difficult toresolve in complex biological fluids,¹⁰ and can be rapidly degraded bothin vivo and ex vivo.^(4,11)

An alternative to endogenous biomarkers is the systemic administrationof exogenous reporter agents to interrogate biological states asdescribed herein. These approaches offer the potential to tailor agentsto exploit host physiology or interface with disease-specific molecularprocesses as alternative indicators of disease. Examples include thepolysaccharide inulin to assess glomerulus filtration rates, FDG-PET tounveil regions of increased glucose metabolism, and a suite of molecularand activity-based probes for imaging biological activities invivo.¹²⁻¹⁴ Because these agents can be designed and tested in vitro andin preclinical models, they can be iteratively optimized and can beadministered at concentrations significantly above biologicalbackground. The limitations with these approaches include the inabilityto monitor large family of probes simultaneously due to limitedmultiplexing capabilities and substantial infrastructure for in vivoanalysis requiring patients to be on-site (e.g. PET, MRI) precludingremote data or sample collection.

Some aspects of this disclosure provide a framework for engineeringnanoscale, isobaric mass-encoded reporter agents that passivelyaccumulate in diseased tissues from host circulation via organ- ordisease-specific vascular fenestrations (e.g. liver sinusoid endotheliumor angiogenic tumor vessels respectively).^(15, 16)In an exemplaryembodiment, iCOREs are provided that are designed to interrogateprotease activity in tumor microenvironments. Upon arrival in thediseased microenvironment, the iCORE agents interface with aberrantlyactive proteases to direct the cleavage and release ofsurface-conjugated, mass-encoded peptide substrates into host urine fordetection by mass spectrometry (MS) as synthetic biomarkers of disease.Because dysregulated protease activities are implicated in a wide rangeof human diseases including cancer, fibrosis, atherosclerosis,inflammation, Alzheimer's and many others,¹⁷ highly multiplexedmonitoring of aberrant protease activities has the potential todistinguish diverse disease states through combinatorial analysis. Whilethe methods and reagents provided herein are widely applicable to a widevariety of diseases, described herein is the exemplary application ofthis technology to address two unmet clinical challenges: the need for anoninvasive alternative to biopsy-based monitoring for liver fibrosis,¹⁸and the inability of current clinically-utilized blood biomarkers toreliably detect early stage cancers.¹⁹

Other exemplary parameters that can be encoded and measured with iCOREtechnology are analytes. Current tools for analyte detection include gelelectrophoresis, western blot, ELISA, PCR, immunofluorescence,microarray, and MS-based platforms like MALDI and LiquidChromatography-MS technologies, such as LC MS/MS. LC MS is an analyticalchemistry technique that combines the physical separation capabilitiesof liquid chromatography (e.g., High Performance LC, HPLC) with the massanalysis capabilities of mass spectrometry. LC-MS has very highsensitivity and selectivity and is, thus broadly applicable to a varietyof analytes, for example, to the specific detection and/oridentification of analytes in the presence of other chemicals (e.g., ina complex biological, experimental, or environmental). MS/MS (or tandemMS) involves two or more steps of MS, with some form of fragmentationoccurring in between the stages. MS/MS is a technique commonly used toidentify sequence information, for example, sequence information ofindividual peptides. Modern mass spectrometers, in-line with extensivechromatographic fractionation, can detect and assess hundreds ofanalytes from a single sample. MS, however, is not without limitations.Current challenges associated with mass spectrometric analysis can besimplified into two major contributing factors.

First, many samples, for example, many biological samples (e.g., blood,serum, tissue (e.g., tumor) biopsy, cell lysate, urine, cerebral spinalfluid), experimental samples (e.g., samples of interest in combinatorialdrug screens), and environmental samples (e.g., soil or water samples)containing or suspected to contain an analyte of interest are highlycomplex, comprising different types of analytes, e.g., biomolecules(e.g., proteins, nucleic acids, lipids, carbohydrates, metabolites),small molecules, drugs and drug metabolites, inorganic and organicmatter, cells, or cell debris, which are present in concentrationsspanning many orders of magnitude (e.g. pg/ml to mg/ml in blood). Thetask of finding and detecting target analytes within this such complexsamples is difficult because of high background signal and moreimportantly, the suppression of target analyte ionization, an integralprocess for mass spectrometric analysis, by bystander molecules (e.g.lipids).

Second, many analytes themselves are difficult to detect reliably androbustly via MS base methods, or, for some analytes, any conventionalmethod for that matter, because of their suboptimal physiochemicalproperties (e.g. mass, charge, hydrophilicity). Variability inphysiochemical properties amongst analytes leads to poor experimentalrepeatability and high variance between assays. This is particularlyrelevant for protein quantification. Often, protein-comprising samplesare digested with a protease, e.g., trypsin, prior to MS analysis, andtypically, the majority of peptides produced from trypsin digests do notcontain sequences that are optimal for MS analysis. Such peptides arenot detected or only poorly detected in a typical MS experiment,resulting in gross under-sampling and decreased overall sensitivity ofcurrent MS assays.

To address these challenges, technologies and methodologies have focusedon affinity enrichment of the target analyte prior to MS to increasesignal intensity, selective depletion of high abundance biomolecules toreduce background, and targeted chemistries to isolate sub-proteomes(see, e.g., Anderson, N. L., Anderson, N. G., Haines, L. R., Hardie, D.B., Olafson, R. W., Pearson, T. W., 2004. Mass spectrometricquantitation of peptides and proteins using Stable Isotope Standards andCapture by Anti-Peptide Antibodies (SISCAPA). J. Proteome Res. 3, 235.;Whiteaker, J. R., Zhao, L., Zhang, H. Y., Feng, L. C., Piening, B. D.,Anderson, L., Paulovich, A. G., 2007b. Antibody-based enrichment ofpeptides on magnetic beads for mass-spectrometry-based quantification ofserum biomarkers. Anal. Biochem. 362, 44; Kuhn, E., Addona, T.,Keshishian, H., Burgess, M., Mani, D. R., Lee, R. T., Sabatine, M. S.,Gerszten, R. E., Carr, S. A., 2009. Developing multiplexed assays forTroponin I and Interleukin-33 in plasma by peptide immunoaffinityenrichment and targeted mass spectrometry. Clin. Chem. 55, 1108;Wollscheid B, Bausch-Fluck D, Henderson C, O'Brien R, Bibel M, SchiessR, Aebersold R, Watts J D., Mass-spectrometric identification andrelative quantification of N-linked cell surface glycoproteins. NatBiotechnol. 2009 April; 27(4):378-86.; Hui Zhang, Xiao-jun Li, Daniel BMartin, Ruedi Aebersold, Identification and quantification of N-linkedglycoproteins using hydrazide chemistry, stable isotope labeling andmass spectrometry. Nature Biotechnology 21, 660-666 (2003); the entirecontents of all of which are incorporated herein by reference). Someefforts have also been directed at developing computational algorithmsto predict high-responding peptides from a given protein, and to reducethe resources required to develop robust proteomic assays (see, e.g.,Mallick, P., Schirle, M., Chen, S. S., Flory, M. R., Lee, H., Martin,D., Ranish, J., Raught, B., Schmitt, R., Werner, T., Kuster, B.,Aebersold, R., Computational prediction of proteotypic peptides forquantitative proteomics. Nat. Biotechnol. 25: 125-131, 2007; Fusaro, V.A., Mani, D. R., Mesirov, J. P., Carr, S. A., Prediction ofhigh-responding peptides for targeted protein assays by massspectrometry. Nat. Biotechnol. 27: 190-198, 2009).

In general, conventional approaches to address the shortcomings ofcurrent MS technology focus on developing novel chemistries andtechnologies to improve quantitative mass analysis, accepting theunderlying condition that certain biological compounds are moredifficult to ionize and detect by virtue of their chemical structure.

Some aspects of this invention, in contrast, provide molecules,compositions, and methods for the translation of target analytes intobiochemical moieties with high ionization efficiency and, thus,optimized for MS-based detection and quantification. In someembodiments, this translation of analyte identity and quantity intobiochemical structures optimized for MS allows for the simultaneousassessment of multiple analytes, for example, analytes of differentphysiochemical properties, and, thus, of different detectability in MSbased assays. In some embodiments, multiple analytes of differentphysiochemical properties are translated into a set of isobaric massreporters, for example, isobaric peptide mass tags that are optimizedfor MS based detection, for example, in MS/MS assays. In someembodiments, the translation allows for the qualitative and/orquantitative analysis of a plurality of target analytes by LC MS/MS. Insome embodiments, biochemical encoding of analytes of differentphysiochemical properties into isobaric mass tags as provided hereinallows to focus the MS analysis on the specific mass window of theisobaric mass tags, which increases sensitivity and accuracy of theanalysis and/or reduces the time required to monitor the relevant masswindow. In some embodiments, the translation comprises a step ofenriching target analytes, for example, target analytes in a complexbiological sample.

Some aspects of this invention provide isotope-encoded reporters(iCOREs), which can be used as mass tags. In some embodiments, iCOREsare provided that are attached to binding agents, e.g., antibodies orantibody fragments, for example, via photo-labile linkers. In someembodiments, iCOREs are used for the qualitative or quantitativedetection of a target analyte by an MS assay, for example, by LC MS/MS.In some embodiments, iCOREs are used for the qualitative or quantitativedetection of an activity, for example, of a target enzyme activity(e.g., a protease, kinase, or phosphatase activity) by an MS assay, forexample, by LC MS/MS.

In some embodiments, complex samples, for example, biological orclinical samples, containing target analytes are first selectivelyenriched by capture antibodies coated onto magnetic microspheres (see,e.g., FIG. 1 ). The immobilized analytes are then contacted withiCORE-labeled binding agents. Following binding and removal of unboundbinding reagents, individual iCOREs are cleaved from the binding agentsthrough UV irradiation and the pool of these “rescued” iCOREs isanalyzed by an MS assay, for example, by LC MS/MS. In some embodiments,the presence and/or abundance of individual iCOREs, as determined by thepresence and/or the signal strength obtained from each iCORE, is used todetermine the presence or absence and/or the abundance (e.g., theconcentration) of a target analyte or activity.

In some embodiments, one or more iCOREs are administered to a subject.For example, in some embodiments, iCOREs may be formulated, administeredto a subject, and collected according to the methods described herein,or those described in PCT Application PCT/US2010/000633, filed on Mar.2, 2010, and entitled Methods And Products For In Vivo Enzyme Profiling,the entire contents of which are incorporated herein by reference. Forexample, iCOREs designed to interrogate analytes or enzyme activity in asubject suspected of having a disease may be administered to thesubject, and analyte- or enzyme-modified iCOREs may be collected at atime sufficient for the iCOREs to be exposed to the analyte or enzymeactivity in the subject. In some embodiments, a sample (e.g., a urine,blood, serum, or plasma sample) is collected from the subject and“rescued” iCOREs are detected within the urine sample, e.g., asdescribed herein or in PCT Application PCT/US2010/000633. In someembodiments, the presence and/or abundance of individual iCOREs, asdetermined by the presence and/or the signal strength obtained from eachiCORE, is used to determine the presence or absence and/or the abundance(e.g., the concentration) of a target analyte or activity.

Some aspects of this invention also provide a method for producing sets,or libraries, of mass codes from a single peptide-based reporter toenable multiplexed experiments in which multiple analytes aresimultaneously detected and/or quantified in a single sample. In someembodiments, an iCORE with favorable MS properties is selected. Somenon-limiting examples of suitable iCOREs are described herein andadditional iCOREs useful according to some aspects of this inventionwill be apparent to those of skill in the art based on this disclosure.For example, in some embodiments, a glu-fib peptide (EGVNDNEEGFFSAR, SEQID NO: 1) is used as the parent iCORE. Starting from this parentsequence, a set of isotopic analogs (e.g., glu-fib iCOREs with uniquefragmentation signature) are designed, thus creating an iCORE librarycomprising a number of iCOREs of identical mass (isobaric iCOREs), butwith distinct fragmentation reporter ions, e.g., y-ions of differentmasses. Such isobaric iCOREs are indistinguishable during MS analysis,but following peptide fragmentation, e.g., by collision induceddisassociation (CID), infrared multiphoton dissociation (IRMPD), or anyother suitable fragmentation method known to those of skill in the art),each member of the set, can be distinguished by its unique fragmentationreporter ion in a tandem MS (MS/MS) assay (FIG. 2 ). The generation ofunique, distinguishable fragmentation signatures is accomplished, insome embodiments, by the strategic substitution of stable-isotopeenriched amino acids in the parent peptide sequence. In someembodiments, a set of binding agents is conjugated to a set of iCOREshaving a set of unique, distinguishable fragmentation signatures, forexample, a set of iCOREs differentially labeled with isotopes that giverise to fragmentation ions that can be identified by MS/MS, so that eachunique isotope-labeling pattern, and associated fragmentation ionsignature, is associated only with binding agents specifically bindingone analyte. In such embodiments, the cognate specificity of eachbinding agent is encoded by the isotope-labeling pattern, and theassociated fragmentation signature of the associated iCORE. In someembodiments, such a set of binding agent-conjugated iCOREs is used forthe multiplexed detection and/or quantification of analytes oractivities in a sample, for example, a biological or clinical sample, orin vivo, according to methods provided herein.

There are several advantages associated with the reagents and methodsprovided herein as compared to conventional MS-based analyte detectionmethodology. For example, in some embodiments, target analytes areenriched by binding agents, e.g., by capture antibodies specificallybinding an analyte of interest, immobilized on a solid substrate, e.g.,a magnetic bead or microsphere, or a membrane or resin. In someembodiments, this enrichment allows for more sensitive and/or specificanalyte detection, and/or reduces background signal, particularly incomplex samples, such as samples of body fluids (e.g. blood or serum),tissues, or cells.

Further, in some embodiments, target analytes are translated intosurrogate isotope-coded reporters (iCOREs) that are pre-designed forfacile detection and/or quantification by MS/MS. In some embodiments,this translation, or biochemical encoding, of analytes, e.g., analyteswhich are challenging to detect by MS assays, into MS tags that arefacile to detect, allows for the circumvention of challenges ofdetecting endogenous chemical structures or peptides directly. This isof particular advantage in the context of multiplexed assays, in which aplurality of analytes of different structure are assayed. Directdetection of different structures in one multiplex MS assay typicallyrequires the screening of a large mass window and while some analytescan readily be detected via MS, many analytes are hard or impossible todetect without extensive pre-processing, or cannot be identifiedunambiguously when analyzing complex samples.

In some embodiments, a single analyte molecule is translated into aplurality of iCORE molecules, resulting in a higher sensitivity ofanalyte detection, for example, by a signal amplification of about 3-20fold. For example, in some embodiments, a binding agent thatspecifically binds a single molecule or two molecules of a targetanalyte, e.g., an antibody or fragment thereof, is conjugated to aplurality of iCORE molecules, for example, to about 3, about 5, about10, or about 20 iCORE molecules, providing an about 3, about 5, about10, or about 20 fold amplification of the number of molecules availablefor MS as compared to direct MS detection of the target analyte,respectively. It will be appreciated by those of skill in the art thathigher amplification rates can be achieved by attaching more iCOREmolecules to a binding agent molecule. Methods and reagents for theattachment of iCOREs to binding agents are provided herein andadditional methods will be apparent to those of skill in the art. Theuse of photo-labile linkers during the translation process, for example,in embodiments where iCOREs bound to binding agents, e.g. antibodies orantibody fragments, are employed, enables ultra-violet light-triggeredrelease of iCOREs from the binding agents. The high efficiency of thisphotochemical process is in marked contrast with the derivation ofpeptides from proteins by enzymatic digestion (necessary for multiplexedprotein detection in conventional LC MS/MS assays) which is limited bythe biophysical properties of the enzyme employed (e.g. KD, kcat,substrate specificity) and the requirement for optimal sample conditionsfor enzymatic activity (e.g. pH, salt concentration). Both of theseconstraints lead to increased sample processing requirements and/ordecreased detection efficiency.

Further, the use of pre-determined mass tags, e.g., isobaric iCOREs,with known masses greatly simplifies the collection and analysis of datasince it is unnecessary to query a large mass window (e.g. 50-2000 m/z)for analytes of diverse molecular weight. Rather, with isobaric codes,narrow mass windows can be centered on the parent mass (e.g. ±0.5 m/z)to efficiently collect signal. In some embodiments, this targetedapproach, combined with analyte signal amplification during translation,increases detection sensitivity by ˜30-300 fold. Moreover, in someembodiments, the simplification of data collection and the resultingdecrease in spectrometer operating time reduces overall costs by ˜10fold.

Further, the method of generating isobaric iCORE mass tag libraries, forexample, isobaric iCORE peptide libraries, is an improvement overcurrent technologies because of the large degree of encoding that can beachieved, which translates into multiplexing capabilities far beyond thecurrent limitations. The current state-of-the-art in isobaric MSmultiplexing is iTRAQ mass tag technology (Ross P L, Huang Y N, MarcheseJ N, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S,Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F,Jacobson A, Pappin D J., Multiplexed protein quantitation inSaccharomyces cerevisiae using amine-reactive isobaric tagging reagents,Mol Cell Proteomics. 2004 December; 3(12):1154-69). ITRAQ labelingreagents are amine-reactive small molecules providing for 4 or maximally8 unique mass codes. A larger library is precluded by the number ofatoms available in small molecules for isotopic substitution. WithiCOREs, e.g., peptide iCOREs, as provided by some aspects of thisinvention, however, the number of atoms available for isotopicsubstitution is increased by the number of monomers, e.g., amino acids,that can be labeled with heavy isotopes. Accordingly, it is feasible toconstruct, for example, at least ˜30-40 unique codes from an averagepeptide length of ˜15 amino acids. The library size, and, thus, thenumber of unique encoding tags can be increased further by using longerpeptides and/or by combining distinct sets of isobaric iCORE peptides,providing for ˜100-1000, or more, elements.

Some aspects of this invention provide isotope-coded reporter molecules(iCORE) and methods of their use. In some embodiments, iCOREs are usefulas mass tags in MS assays, for example, in multiplex LC MS/MS assays, asdescribed in more detail elsewhere herein. In some embodiments, iCOREsare useful in the simultaneous qualitative and/or quantitative detectionof tens, hundreds, or thousands of analytes in a sample, for example, acomplex biological sample, or in the simultaneous tracking of tens,hundreds, or thousands of cells, tissues, or samples using an MSreadout.

In some embodiments, a set, plurality, or library of different iCOREs isprovided that is useful in multiplex MS assays, for example, inmultiplex LC MS/MS assays. In some embodiments, the iCOREs of a set,plurality, or library of iCOREs are isobaric. For example, in someembodiments, a set, plurality, or library of isobaric iCOREs is providedin which all iCOREs are polymers, e.g., peptides of the same amino acidsequence (e.g., glu-fib peptides (EGVNDNEEGFFSAR, SEQ ID NO: 1)), andhave, accordingly, the same physiochemical properties as relevant for MSanalysis. Accordingly, all iCOREs of such a set, plurality, or libraryare detected with substantially the same specificity, sensitivity, andaccuracy in an LC MS/MS assay. In some embodiments, different iCOREs insuch a set, plurality, or library of isobaric iCOREs have differentfragmentation signatures, which can readily be distinguished in an MS/MSassay as described in more detail elsewhere herein. Because even shortpolymers, e.g., short polypeptides, allow for the generation of a largenumber of unique fragmentation signatures in a set, plurality or libraryof isobaric iCOREs that can be distinguished via MS/MS assays, forexample, by differential isotope-labeling of the iCOREs, this technologycan be used for multiplex qualitative or quantitative MS analysis farbeyond the limitations of current multiplex MS technologies, such asiTRAQ (4- or 8-plex max.) or tandem mass tag (TMT, 6-plex max.)technologies.

A non-limiting, exemplary set of 10 isobaric peptide iCOREs of the aminoacid sequence EGVNDNEEGFFSAR (SEQ ID NO: 1) is described in FIG. 3 . Thedifferent iCOREs G1-G10 have different isotope patterns, and, thus,different fragmentation signatures, e.g., G1 has the isotope patternE⁺³G₊₆VNDNEEGFFSAR (SEQ ID NO: 1), G2 has the isotope patternE⁺²G⁺⁶VNDNEE⁺¹GFFSAR (SEQ ID NO: 1), G3 has the isotope patternE⁺¹G⁺⁶VNDNEE⁺²GFFSAR (SEQ ID NO: 1), and so forth. In the first round ofMS, only a single peak is observed, reflecting the isobaric nature ofiCOREs G1-10 (FIG. 3 I.). After fragmentation, however, the uniquefragmentation signatures of G1-10 result in resolution of the fragmentsas 10 distinct peaks (FIG. 3 I.). The lower panel of the figure shows aclose-up of the peaks obtained from the y7 ion (EGFFSAR, SEQ ID NO: 6,see FIG. 2 for y ion nomenclature), allowing qualitative(present/absent) as well as quantitative analysis of each unique iCOREin the set. Those of skill in the art will appreciate that the glu-fibpeptide sequence allows for the generation of more than 10 uniquefragmentation signatures via differential isotope labeling, for example,by further encoding the remaining amino acids within the sequence, andthat the exemplary set of ten iCOREs is not limiting in this respect.The multiplexing capabilities of iCORE technology are limited only bythe number of unique fragmentation patterns that can be created iniCOREs. Even a small peptide sequence, such as the glu-fib peptidesequence exemplified in FIG. 3 allows for the generation of at least30-40 unique isotope signatures, if commercially availableisotope-labeled amino acids are used, and more if such amino acids arecustom-synthesized.

Three non-limiting, exemplary differential isotope-labeling strategiesfor generating multiplex glu-fib based iCORE libraries are providedbelow. In these examples, iCOREs are isotope-labeled by substitution ofone or more atoms in the indicated amino acids with one or more heavyisotopes, resulting in a change in mass of the labeled amino acid. Theresulting iCOREs in each library are isobaric, with different iCOREshaving a different fragmentation signature, e.g., with respect to the y₇ion. The examples are intended for illustration only and do not limitthis aspect of the invention. Additional useful differentialisotope-labeling strategies and schemes will be apparent to those ofskill in the art.

10-Plex Glu-Fib iCORE Library (G₁₀):

G₁₀1=E-⁺³G-⁺⁶V-N-D-N-E-E-G-F-F-S-A-R

G₁₀2=E-⁺²G-⁺⁶V-N-D-N-E-E-⁺¹G-F-F-S-A-R

G₁₀3=E-⁺¹G-⁺⁶V-N-D-N-E-E-⁺²G-F-F-S-A-R

G₁₀4=E-G-⁺⁶V-N-D-N-E-E-⁺²G-F-F-S-⁺¹A-R

G₁₀5=E-G-⁺⁵V-N-D-N-E-E-G-F-F-S-⁺⁴A-R

G₁₀6=E-⁺³G-⁺¹V-N-D-N-E-E-⁺¹G-F-F-S-⁺⁴A-R

G₁₀7=E-⁺³G-V-N-D-N-E-E-G-⁺⁶F-F-S-A-R

G₁₀8=E-⁺²G-V-N-D-N-E-E-G-⁺⁶F-F-S-⁺¹A-R

G₁₀9=E-⁺¹G-V-N-D-N-E-E-⁺²G-⁺⁶F-F-S-A-R

G₁₀10=E-G-V-N-D-N-E-E-⁺³G-⁺⁶F-F-S-A-R   (SEQ ID NO: 36)

18-Plex Glu-Fib iCORE Library (G₁₈)

G₁₈1=E-⁺³G-⁺⁶V-⁺⁶N-D-⁺²N-E-E-G-F-F-S-A-R

G₁₈2=E-⁺²G-⁺⁶V-⁺⁶N-D-⁺²N-E-E-G-F-F-S-⁺¹A-R

G₁₈3=E-⁺¹G-⁺⁶V-⁺⁶N-D-⁺²N-E-E-⁺¹G-F-F-S-⁺¹A-R

G₁₈4=E-G-⁺⁶V-⁺⁶N-D-⁺²N-E-E-⁺²G-F-F-S-⁺¹A-R

G₁₈5=E-G-⁺⁵V-⁺⁶N-D-⁺²N-E-E-G-F-F-S-⁺⁴A-R

G₁₈6=E-⁺¹G-⁺⁵V-⁺⁶N-D-N-E-E-⁺¹G-F-F-S-⁺⁴A-R

G₁₈7=E-⁺³G-V-⁺⁶N-D-⁺²N-E-E-G-⁺⁶F-F-S-A-R

G₁₈8=E-⁺²G-V-⁺⁶N-D-⁺²N-E-E-G-⁺⁶F-F-S-⁺¹A-R

G₁₈9=E-⁺¹G-V-⁺⁶N-D-⁺²N-E-E-⁺¹G-⁺⁶F-F-S-⁺¹A-R

G₁₈10=E-⁺¹G-⁺⁵V-N-D-⁺²N-E-E-⁺²G-⁺⁶F-F-S-⁺¹A-R

G₁₈11=E-G-⁺⁵V-N-D-⁺²N-E-E-G-⁺¹⁰F-F-S-A-R

G₁₈12=E-⁺³G-⁺¹V-N-D-⁺²N-E-E-G-⁺¹⁰F-F-S-⁺¹A-R

G₁₈13=E-⁺²G-⁺¹V-N-D-+2N-E-E-⁺¹G-⁺¹⁰E-F-S-⁺¹A-R

G₁₈14=E-⁺²G-V-N-D-⁺²N-E-E-⁺²G-⁺¹⁰F-F-S-⁺¹A-R

G₁₈15=E-⁺¹G-V-N-D-⁺²N-E-E-G-⁺¹⁰F-F-S-⁺⁴A-R

G₁₈16=E-⁺²G-V-N-D-N-E-E-⁺¹G⁺¹⁰F-F-S-⁺⁴A-R

G₁₈17=E-⁺¹G-V-N-D-N-E-E-G⁺¹⁰F-⁺⁶F-S-A-R

G₁₈18=E-G-V-N-D-N-E-E-G-⁺¹⁰F-⁺⁶F-S-⁺¹A-R   (SEQ ID NO: 36)

22-Plex Glu-Fib iCORE Library (G₂₂)

G₂₂1=E-⁺³G-⁺⁶V-⁺⁶N-D-⁺⁶N-E-E-G-F-F-S-A-R

G₂₂2=E-⁺²G-⁺⁶V-⁺⁶N-D-⁺⁶N-E-E-G-F-F-S-⁺¹A-R

G₂₂3=E-⁺¹G-⁺⁶V-⁺⁶N-D-⁺⁶N-E-E-⁺¹G-F-F-S-⁺¹A-R

G₂₂4=E-G-⁺⁶V-⁺⁶N-D-⁺⁶N-E-E-⁺²G-F-F-S-⁺¹A-R

G₂₂5=E-G-⁺⁵V-⁺⁶N-D-⁺⁶N-E-E-G-F-F-S-⁺⁴A-R

G₂₂6=E-G-⁺⁵V-⁺⁶N-D-⁺⁶N-E-E-⁺¹G-F-F-S-⁺⁴A-R

G₂₂7=E-⁺³G-V-⁺⁶N-D-⁺⁶N-E-E-G-⁺⁶F-F-S-A-R

G₂₂8=E-⁺²G-V-⁺⁶N-D-⁺⁶N-E-E-G-⁺⁶F-F-S-⁺¹A-R

G₂₂9=E-⁺¹G-V-⁺⁶N-D-⁺⁶N-E-E-⁺¹G-⁺⁶F-F-S-⁺¹A-R

G₂₂10=E-⁺¹G-⁺⁵V-N-D-⁺⁶N-E-E-⁺²G-⁺⁶F-F-S-⁺¹A-R

G₂₂11=E-G-⁺⁵V-N-D-⁺⁶N-E-E-G-⁺¹⁰F-F-S-A-R

G₂₂12=E-⁺³G-⁺¹V-N-D-⁺⁶N-E-E-G-⁺¹⁰F-F-S-⁺¹A-R

G₂₂13=E-⁺²G-⁺¹V-N-D-⁺⁶N-E-E-⁺¹G-⁺¹⁰F-F-S-⁺¹A-R

G₂₂14=E-⁺²G-V-N-D-⁺⁶N-E-E-⁺²G-⁺¹⁰F-F-S-⁺¹A-R

G₂₂15=E-⁺¹G-V-N-D-⁺⁶N-E-E-G-⁺¹⁰F-F-S-⁺⁴A-R

G₂₂16=E-⁺¹G-⁺⁵V-N-D-N-E-E-⁺¹G-⁺¹⁰F-F-S-⁺⁴A-R

G₂₂17=E-G-⁺⁵V-N-D-N-E-E-G-⁺¹⁰F-⁺⁶F-S-A-R

G₂₂18=E-⁺³G⁺¹V-N-D-N-E-E-G-⁺¹⁰F-⁺⁶F-S-⁺¹A-R

G₂₂19=E-⁺²G-⁺¹V-N-D-N-E-E-⁺¹G-⁺¹⁰F-⁺⁶F-S-⁺¹A-R

G₂₂20=E-⁺²G-V-N-D-N-E-E-⁺²G-⁺¹⁰F-⁺⁶F-S-⁺¹A-R

G₂₂21=E-⁺¹G-V-N-D-N-E-E-G-⁺¹⁰F-⁺¹⁰F-S-A-R

G₂₂22=E-G-V-N-D-N-E-E-G-⁺¹⁰F-⁺¹⁰F-S-⁺¹A-R   (SEQ ID NO: 36)

In some embodiments, the isotope-labeled amino acids are D-amino acids.In some embodiments, the isotope-labeled amino acids are L-amino acids.In some embodiments, the isotope-labeled amino acids are a mix of D- andL-amino acids.

Those of skill in the art will appreciate that longer polymers, forexample, longer polypeptides, polysaccharides, or polynucleotides, allowfor even more unique fragmentation signatures to be generated bydifferential isotope labeling, thus further expanding the multiplexingcapabilities of iCORE technology.

In some embodiments, a set, plurality, or library of iCOREs (e.g.polypeptide, polynucleotide, or polysaccharide iCOREs) is provided thatcomprises at least 2, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 25, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 125, at least 150, at least 200, at least 250, at least 500, atleast 1000, or more than 1000 different iCOREs, e.g. iCOREs that have aunique fragmentation signature that is distinguishable by an MS assay,e.g. an MS/MS assay. In some embodiments, the iCOREs in the set,plurality, or library are isobaric iCOREs, for example, isobaricpolypeptide, polynucleotide, or polysaccharide iCOREs. In someembodiments, each unique fragmentation signature in a set of iCOREs, forexample, a set of isobaric iCOREs, is associated with, or represents, aspecific analyte, or parameter. Methods for the use of iCOREs, forexample, in analyte detection, as well as cell, tissue, sample, andliquid tracking, are also provided.

The term iCORE refers to isotope-coded reporter molecules. In someembodiments, an iCORE is a molecule that is readily detectable by MStechnology. In some embodiments, an iCORE is a peptide, for example, apeptide known to be readily detectable by MS technology. In otherembodiments, an iCORE is a polynucleotide or a polysaccharide.Typically, an iCORE is a molecule that allows for differentialisotope-labeling, e.g., for the generation of unique fragmentationsignatures that are distinguishable in a detection assay, such as MS/MS.This allows for the generation of libraries of isobaric iCOREs whichshare the same physiochemical properties for the purpose of MSdetection, for example, peptide iCOREs having the same amino acidsequence, and are readily distinguishable by their unique fragmentationsignature, for example, in LC MS/MS assays. Typically, an iCORE is apolymer comprised of monomers that exhibit different MS signatures and,thus, comprises a sequence of monomers. Such polymers are well known tothose of skill in the art and include, but are not limited to polymersof amino acids, nucleotides, and monosaccharides, for example,polypeptides, polynucleotides, and polysaccharides, respectively.

The length of iCOREs is typically chosen to produce a mass signaturethat is useful in MS assays, for example, a mass signature that canreadily be detected in an MS/MS assay. For example, in some embodiments,an iCORE is a polymeric molecule (e.g., a polypeptide, polynucleotide,or polysaccharide) that comprises at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 25, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, or at least 100monomer residues, for example, amino acid, nucleotide, or monosaccharideresidues. In some embodiments, an iCORE is a polymeric molecule, forexample, a polypeptide, polynucleotide, or polysaccharide, thatcomprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51-60, 61-70, 71-80, 81-90, or91-100 monomers, e.g., amino acid or nucleotide residues. In someembodiments, an iCORE is a polymer comprising more than 100 monomers,for example, more than 100 amino acid, nucleotide, or monosaccharideresidues.

Non-limiting examples of polymeric molecules that are useful for thegeneration of iCOREs are the polypeptides glu-fib (EGVNDNEEGFFSAR (SEQID NO: 1)), bradykinin (PPGFSPFR (SEQ ID NO: 30)), angiotensin I(DRVYIHPFHL (SEQ ID NO: 31)), ACTH1-17 (SYSMEHFRWGKPVGKKR (SEQ ID NO:32)), ACTH18-39 (RPVKVYPNGAEDESAEAFPLEF (SEQ ID NO: 33)), and ACTH7-38(FRWGKPVGKKRRPVKVYPNGAEDESAEAFPLE (SEQ ID NO: 34)). Additional polymericmolecules and sequences that can be used for the generation of iCOREswill be readily evident to those of skill in the art and the inventionis not limited in this respect.

In order to generate an iCORE from a non-labeled polymeric molecule, forexample, a polypeptide, polynucleotide, or polysaccharide, the polymericmolecule is isotope-labeled. Methods and reagents for isotope-labelingof polymeric molecules are well known to those of skill in the art. Insome embodiments, such methods include the generation of a polymericmolecule, for example, a peptide, from monomers, for example, aminoacids, at least some of which are isotope-labeled to obtain anisotope-labeled polymeric molecule. In some embodiments,isotope-labeling is effected by the introduction of heavy isotopes.Isotope-labeling with radioactive isotopes or with stable isotopes ispossible. In some embodiments, labeling with stable isotopes ispreferred.

For example, while non-labeled monomers, e.g., non-labeled amino acids,nucleotides, or monosaccharides, predominantly comprise C¹², O¹⁶, N¹⁴,and S³² atoms, isotope-labeled monomers, e.g., isotope-labeled aminoacids, may comprise one or more heavy isotope(s), for example, a C¹³,N¹⁵, O¹⁷, O¹⁸, H², S³³, S³⁴, or S³⁶ isotope. A monomer comprising aheavy isotope has a different mass than a non-labeled amino acid, andeven a single isotope comprised in a monomer can readily be measured byMS assays. This difference in mass is conferred to any polymercomprising such an isotope-labeled monomer. For example, the differencein mass resulting from the substitution of a C¹² atom of an amino acidwith a C¹³ isotope can be measured in a peptide comprising such anisotope-labeled amino acid.

To give a non-limiting example of monomer isotope labeling, the phenylring of non-labeled phenylalanine (F) typically comprises six C¹² atoms.In some embodiments, an isotope-labeled phenylalanine residue comprisesa phenyl ring comprising five C¹² atoms and one C¹³ isotope, increasingthe mass of the labeled amino acid by the mass of one neutron (⁺¹F). Insome embodiments, an isotope-labeled phenylalanine residue comprises aphenyl ring comprising four C¹² atoms and two C¹³ isotopes, increasingthe mass of the labeled amino acid by the mass of two neutrons (⁺²F). Insome embodiments, an isotope-labeled phenylalanine residue comprises aphenyl ring comprising three C¹² atoms and three C¹³ isotopes (⁺³F), twoC¹² atoms and four C¹³ isotopes (⁺⁴F), one C¹² atoms and five C¹³isotopes (⁺⁵F), or six C¹³ isotopes (⁺⁶F). The remaining three C12 atomsof non-labeled phenylalanine and as well as the N14 atom of the aminogroup can also be isotope-substituted, yielding ⁺⁷F, ⁺⁸F, ⁺⁹F, and ⁺¹⁰F.It will be appreciated by those of skill in the art that any combinationof isotope substitutions are possible, for example, substitution of theN¹⁴ of the amino group, of the C¹² of the carboxy group and of two C¹²of the phenyl ring would produce ⁺⁴F, as would a substitution of threephenyl ring C¹² with C¹³ and of the N¹⁴ with N¹⁵, or substitution offour C¹² of the phenyl ring with 4 C¹³. In some embodiments, theisotope-labeled phenylalanine is used to generate an isotope-labeledpeptide. In some embodiments, this involves peptide synthesis in vivo,for example, by cells that are incubated in the presence ofisotope-labeled phenylalanine and incorporate the labeled amino acidinto any peptide they synthesize. In some embodiments, isotope-labeledpeptides are synthesized in vitro, for example, via fmoc synthesis usingisotope-labeled amino acids as building blocks. It will be appreciatedby those of skill in the art that amino acids other than phenylalaninecan be isotope-labeled according using similar strategies and methodsand reagents known to those of skill in the art and the invention is notlimited in this respect. It will further be appreciated that monomersother than amino acids can be isotope-labeled, for example, nucleotidesand monosaccharides. Further, other methods for the generation ofisotope-labeled polymers that are useful as iCOREs, for example, methodsin which a polymer is directly labeled, are also known to those of skillin the art, and the invention is not limited in this respect.

Different fragmentation signatures can be created by differentialisotope labeling of polymeric molecules, for example, by addingdifferent amounts of heavy isotopes to the same monomeric residue, or bygenerating different combinations of labeled monomeric residues withinthe polymeric molecule. For an example of different iCORE fragmentationsignatures created by differential isotope labeling, see iCOREs G1-G10described in FIG. 3 . Isotope-labeling of a polypeptide, polynucleotide,or polysaccharide at a specific residue, for example, a specific aminoacid, nucleotide, or monosaccharide residue, produces a specific isotopelabeling signature that can readily be identified, and distinguishedfrom other polypeptides, polynucleotides, or polysaccharides of the samemonomeric sequence that are labeled at a different residue, via MSassays, for example, via MS/MS assays. This allows for the generation ofmultiple iCOREs comprising the same monomeric sequence, for example,multiple polynucleotide iCOREs comprising the same amino acid sequence,but isotope-labeled at different residues. Such differentially labeled,isobaric iCOREs exhibit identical physiochemical properties for thepurpose of MS assays, and are, thus, detectable at an equal level ofsensitivity, specificity, and accuracy in MS assays, while still beingdistinguishable by their unique fragmentation signature, for example, inLC MS/MS assays as described herein.

In some embodiments, iCOREs are polypeptides. Typically, polypeptideiCOREs comprise a sequence that is optimized for facile detection in MSassays, as exemplified by the iCORE sequences provided herein. In someembodiments, a library of isobaric iCOREs is provided, in which alliCOREs comprise the same amino acid sequence, but different iCOREscomprise different isotope-labeling signatures. Even short polypeptides,e.g. of about 5-20 amino acids in length, allow for the generation oftens or hundreds of unique isotope-labeling signatures, which translatesinto the possibility to detect and analyze tens or hundreds of analytesin a multiplexed MS assay using isobaric iCOREs of such lengths. In someembodiments, a library of peptide iCOREs is provided that comprisesiCOREs of different amino acid sequences, further increasing the numberof unique isotope-labeling signatures and, thus, the number of analytesthat can be assayed in a single multiplexed MS experiment. Suchheterogeneous iCORE libraries can be generated, for example, bycombining a first iCORE library comprising isobaric, differentiallylabeled iCOREs with an additional library comprising isobaric,differentially labeled iCOREs. For example, such a library could begenerated by combining a library of glu-fib based iCOREs with a libraryof bradykinin based iCOREs and a library of angiotensin I-based iCOREs,thus creating a larger, combinatorial library of iCOREs.

Some aspects of this invention relate to the recognition that even arelatively small number of relatively short polypeptide tags that areuseful in MS assays provide virtually unlimited multiplexingcapabilities, since polypeptides are comprised of amino acid residues,and there are 20 naturally occurring and a large number of artificialamino acids that are useful as building blocks for iCOREs. For example,an iCORE library based on isobaric, differentially labeled glu-fib(EGVNDNEEGFFSAR (SEQ ID NO: 1)) iCOREs allows for the detection of about10-100 analytes in a multiplexed MS assay, while an iCORE library basedon isobaric, differentially labeled ACTH7-38(FRWGKPVGKKRRPVKVYPNGAEDESAEAFPLE (SEQ ID NO: 34)) iCOREs allows for thedetection of about 10-400 analytes in a multiplex MS assay.Combinatorial iCORE libraries, accordingly, allow for the assessment ofthousands of analytes simultaneously.

Some aspects of this invention provide sets or libraries of iCOREs. Insome embodiments, the iCOREs comprise or are conjugated to a reactivemoiety that reacts with another molecule. For example, in someembodiments, the iCOREs comprise a reactive moiety that forms a covalentbond to a molecule of interest, for example, to an analyte, or a set ofanalytes. In some embodiments, the molecule of interest is a peptide orprotein, a carbohydrate, a nucleic acid, or a lipid. In someembodiments, the molecule of interest to which the iCORE is aimed to beconjugated is a binding agent, for example, an antibody, antibodyfragment, aptamer, ligand, receptor, or adnectin. For example, in someembodiments, a set of iCOREs is provided that is conjugated to areactive chemical moiety that forms a covalent bond to a protein ifcontacted with a protein. Reactive chemical moieties are well known tothose of skill in the art. One exemplary embodiment is depicted in FIG.7 to illustrate this point. Here, the iCORE is a glu-fib peptide,covalently bound to a chemical moiety that polymerizes under UV lightwith PEG monomers to form a PEG hydrogel. Polyethylene glycol (PEG),also known as poly(oxyethylene) glycol, is a condensation polymer ofethylene oxide and water having the general chemical formulaHO(CH₂CH₂O)[n]H. Such hydrogels are useful, for example, in thegeneration of engineered tissues and the conjugation of an iCORE to sucha hydrogel allows for tracking a tissue grown on such a tagged hydrogelin in vitro or in vivo experiments as described in more detail elsewhereherein. Other reactive chemical moieties and methods for conjugation ofsuch moieties to iCOREs provided herein are well known to those of skillin the art and the invention is not limited in this respect. It will beapparent to those of skill in the art that the type of reactive chemicalmoiety can be selected to be reactive with a moiety comprised in themolecule of interest to which the iCORE is aimed to be conjugated. Insome embodiments, iCOREs are provided that are conjugated to a reactivechemical moiety via a covalent bond. In some embodiments, iCOREs areprovided that are conjugated to a reactive chemical moiety via acleavable linker, for example, a photocleavable linker.

Providing iCOREs conjugated to reactive chemical moieties allows for thecustomized labeling of molecules of interest by the end-user, forexample, by a scientist, thus extending the versatility of iCOREtechnology to custom-made experimental designs. For example, in someembodiments, iCOREs conjugated with a reactive moiety that forms acovalent bond with peptides may be used to label proteins or peptidesobtained from a biological sample for comparison with proteins orpeptides obtained from a different sample, much in the same way as ITRAQtags are commonly used. In contrast to ITRAQ technology, the virtuallyunlimited number of different fragmentation signatures available for agiven iCORE set, for example, a set of isobaric peptide iCOREs asdescribed herein, allows for the simultaneous assessment of a virtuallyunlimited number of analytes, for example, for the simultaneousassessment and comparison of a virtually unlimited number of protein orpeptide samples.

Scalability of iCORE technology depends on the specific binding agentsused (for embodiments, in which iCOREs conjugated to specific bindingagents are used or provided) and the specific, distinguishable massreporters, e.g., iCOREs, having a unique, distinguishable fragmentationsignature. Regarding specific binding agents, iCOREs can be conjugatedto virtually all commercially available binding agents, including, butnot limited to, antibodies, antibody fragments, aptamers, ligand-bindingproteins or proteins domains, and adnectins by methods well known tothose of skill in the art. The number of iCOREs with uniquefragmentation signatures scales with the peptide space, which scales as20^(n), where n=length of peptide, for peptide iCOREs comprising onlynaturally occurring amino acids. Accordingly, even very short peptideiCOREs provide a large number of possible unique fragmentationsignatures, while longer peptides provide virtually limitless uniquesignatures. In some embodiments, peptide iCOREs that are readilydetected by MS are preferable, but even the exemplary, readilydetectable peptide iCOREs described herein alone allow for hundreds orthousands of unique fragmentation signatures. Some peptide sequencessuitable for the generation of iCOREs are provided herein and additionalsuitable peptide sequences will be apparent to those of skill in the artbased on this disclosure.

In some embodiments, iCOREs are provided that are conjugated to abinding agent. A binding agent is an agent that specifically binds amolecule, for example, an analyte. In some embodiments, the bindingagent is an antibody or antibody fragment. In some embodiments, thebinding agent is a peptide or protein. In some embodiments, the bindingagent is an aptamer or adnectin. In some embodiments, the binding agentis a ligand or a receptor, or comprises a ligand binding domain.

Accordingly, in some embodiments, iCORE-binding agent conjugates areprovided that specifically bind to an analyte. In some embodiments, theconjugation of iCORE and binding agent is via a covalent bond, forexample, a covalent peptide bond in an iCORE-binding agent fusionprotein. In some embodiments, the conjugation of iCORE and binding agentis via a cleavable linker, for example, a protease cleavable linker or aphotocleavable linker. In some embodiments, the linker is a peptidelinker comprising a protease cleavage site. Some exemplaryphotocleavable linkers and protease cleavage sites are described herein,and additional linkers and cleavage sites will be apparent to those ofskill in the art, and the invention is not limited in this respect. Forexample, in some embodiments, an iCORE is provided that is conjugated toa binding agent, for example, an antibody, via a photocleavable linker,as described herein. In some embodiments, a set of iCOREs is providedthat is conjugated to a set of binding agents, for example, a set ofantibodies, or antibody fragments, or ligand binding peptides andproteins, or aptamers or adnectins, or any combination of such agents.Typically, the iCOREs in such a set are conjugated to the binding agentsin a manner that allows for the identification of a particular bindingagent, and, thus, the analyte bound by a binding agent, by determiningthe identity of the iCORE.

For example, in a set of 10 different iCOREs (e.g., iCORE G1-G10 in FIG.2 ) that are conjugated to a set of 10 different binding agents(B1-B10), which, in turn, specifically bind a set of analytes (A1-A10),the iCOREs are conjugated to the binding agents so that each iCORE of aunique fragmentation signature is conjugated to particular bindingagent, e.g. G1 to B1, G2 to B2, G3 to B3, and so forth. The uniquefragmentation signature of each iCORE can be identified anddistinguished from the fragmentation signatures of the other iCOREs inthe library in an MS assay, e.g. the MS/MS assay depicted in FIG. 3 .Accordingly, the presence or absence of a specific binding agent in asample can be inferred from the presence or absence of the uniquefragmentation signature of the iCORE associated with that particularbinding agent. In some embodiments, a sample is assayed for the presenceor absence of a set of analytes by contacting the sample with the set ofiCOREs conjugated to the binding agents under conditions suitable forthe binding agents to bind to their respective analytes. Subsequently,those iCORE-conjugated binding agents that are specifically bound totheir respective analytes are enriched or isolated. In some embodiments,the iCOREs are then released, for example, by cleaving the linkerconnecting the iCORE to the binding agent. In some embodiments, theiCOREs are then subjected to MS analysis, for example, in an MS/MSassay, and the presence or absence, and/or the quantity of each iCORE isdetermined. In some embodiments, the presence or absence, and/or thequantity of each analyte is determined from the result of the MS assay.For example, in some embodiments, if an iCORE MS assay results in thedetection of iCOREs G1, G3, G5-7 and G10, the presence of analytes A1,A3, A5-7, and A10, and/or the absence of analytes A2, A4-6, and A8-9 canbe determined. In some embodiments, a comparison of the signal obtainedfor a given iCORE in the MS assay can be used to determine the quantityof the respective analyte in the sample, and/or to compare the level ofthe respective analytes in the sample to the level of a differentanalyte in the sample via MS signal comparison. For example, if the MSresult obtained from a sample contacted with 10 iCOREs conjugated to 10binding agents specifically binding to 10 analytes would represent theMS result as shown in FIG. 3 , the presence of all unique fragmentationsignatures of iCOREs G1-10 would indicate that all analytes A1-10 arepresent in the sample. In some embodiments, the similar signal level ofall unique fragmentation signatures of iCOREs G1-10 may further indicatethat all analytes A1-10 are present at similar levels in the sample, ifthe binding agent-analyte interactions are substantially similar acrossthe analytes.

In some embodiments, a library of isobaric iCOREs is provided in whichthe iCOREs comprise the same sequence, for example, a set of peptide ornucleic acid iCOREs that comprise the same amino acid or nucleotidesequence, respectively, but in which the different iCOREs areisotope-labeled at different positions, for example, at different aminoacid or nucleotide residues within the amino acid or nucleotidesequence.

Some aspects of iCORE technology rely on or use proven, existingtechnologies, for example, bead-based immuno-enrichment, photo-labilebioconjugation chemistry, direct labeling of analytes, for example, ofproteins by reactive chemical moiety conjugation, and qualitative and/orquantitative mass spectrometry, for example, LC MS/MS assays. Thesetechnologies are well known to those of skill in the art and manyvariations and equivalents of the exemplary embodiments described hereinwill be apparent to the skilled artisan based on this disclosure. Itwill be appreciated that iCORE technology can be applied to manydifferent analyte detection scenarios, e.g. diagnostic scenarios, aswell as many different tracking scenarios. The invention is not limitedin this respect.

For example, some embodiments of iCORE technology as described hereinprovide molecular diagnostic assays (e.g. biomarker detection,monitoring of vaccines, and HLA screening) as well as qualitative andquantitative assays for basic science research (e.g. protein expressionprofiling, cell profiling, tissue profiling, and metabolic profiling).For example, certain embodiments provide a library of iCORE-labeledbinding agents (e.g. antibodies or antibody fragments) targeted againsta panel of serum biomarkers (e.g. organ- and/or disease-specificmarkers) that can be used for monitoring biological processes byanalyzing body fluid or tissue samples obtained from a subject (e.g.tumor evolution, host responses after drug administration, or drugmetabolism). Some embodiments of iCORE technology are particularlysuited for such multi-parameter studies, particularly in the diagnosisof diseases or conditions, because iCORE technology allows for increasesin detection sensitivity and resolution through multiplexing, which, inturn, allows for the efficient detection of multi-parameter diagnosticsignatures.

Some embodiments provide a library of chemically-active iCOREs, forexample, isobaric peptide iCOREs conjugated to a reactive chemicalmoiety that can be utilized by an end user to encode user-definedexperiments. For example, in case-and-control comparative proteomicsstudies, proteins from each unique condition, experiment, or subject,can be encoded (e.g., labeled) with a particular iCORE fragmentationsignature. All samples can then be combined and simultaneously analyzedby LC MS/MS to determine the relative effects of the experimentalconditions. The use of iCORE technology enables comparative studies at ascale precluded by current technologies and methods.

It will be appreciated by the skilled artisan that the use of iCOREtechnology, as described herein, is not limited to analyte detection,encoding and translation. Because iCOREs are versatile, novel MS tagsthat have multiple advantages over previously known MS tags, iCOREtechnology is amenable to adaptation to various MS tag detectiontechnologies and diagnostic strategies. For example, iCORE technologycan be used in the context of multiplexed enzyme (e.g., protease)activity profiling using MS/MS strategies, as described in PCTapplication PCT/US2010/000633, published as WO/2010/101628 on Oct. 9,2010, the entire contents of which are incorporated herein by reference.

For example, iCOREs can be conjugated to a carrier, for example, ananoparticle (e.g., a nanoworm (NW)), via a linker comprising an aminoacid sequence that is cleaved by an enzyme of interest, e.g., by aprotease associated with a disease. Such iCORE NWs, also sometimesreferred to as pro-diagnostic reagents, can then be administered to asubject to interrogate the activity of the enzyme in the subject. Insome embodiments, a set of iCOREs is provided that is linked to acarrier via different linkers targeted by different enzymes (e.g.,different proteases) in a way that the activity of each proteasereleases one specific iCORE tag. Used in this manner, iCORE technologycan be employed to interrogate the activities of a plurality ofdisease-associated enzymes (e.g., disease-associated proteases) inparallel in one multiplexed MS assay.

In some embodiments, a set of pro-diagnostic iCORE reagents isadministered to a subject suspected to exhibit an aberrant (e.g.,pathogenic) level of activity of one or more enzymes (e.g., one or moreproteases) to probe the activity levels of numerous proteases in onemultiplexed assay. While the carrier of the prodiagnostic reagent istypically not secreted into the urine of the subject, when exposed toprotease activity, the respective iCORE is cleaved from the NW andreleased into the urine of the subject. Urine samples are collectedafter a sufficient time after administration of the pro-diagnosticreagent to the subject has passed for the reagent to be exposed toenzyme activity in the subject. Urine samples are then subjected toMS/MS assays as described in more detail elsewhere herein, or inPCT/US2010/000633, published as WO/2010/101628, to detect the cleavediCOREs and determine whether or not the subject exhibits an aberrantprotease activity signature.

Protease activities that are associated with disease are well known tothose of skill in the art (see, e.g., Table 1 of PCT/US2010/000633,published as WO/2010/101628, for some non-limiting examples ofdisease-associated enzyme activities, incorporated herein in itsentirety by reference). Protease target sequences that are useful aslinker sequences for iCOREs to carriers are also well known to those ofskill in the art. Some exemplary iCOREs, protease-cleavable linkers, andmethods for the diagnosis of diseases in a subject are described indetail herein. Additional iCOREs, linkers, and methods will be apparentto those of skill in the art based on this disclosure, and thedisclosure is not limited in these aspects. As will be apparent to theskilled artisan, the use of iCORE technology in the context of multiplexprotease activity assays allows for an improvement of multiplexingcapabilities and increased ease of detection as compared to previouslydescribed methods.

Some embodiments provide kits of reagents described herein. For example,some embodiments provide a kit comprising a set of iCOREs, for example,isobaric peptide iCOREs, that comprises iCOREs having differentfragmentation signatures. In some embodiments, the iCOREs are conjugatedto a reactive chemical moiety, for example, a peptide-reactive moietythat forms a covalent bond to a peptide when contacted with such apeptide. In some embodiments, the different iCOREs in a set of iCOREs soprovided are separated, thus allowing the end-user to label a particularsample, for example, a protein sample, with a specific iCORE and adifferent sample with a different iCORE, thus allowing subsequent mixingand simultaneous analysis if the samples in an MS assay, e.g. in an LCMS/MS assay.

In some embodiments, a kit is provided that comprises a set or libraryof binding agents that are conjugated to iCOREs, as described herein. Insome embodiments, each binding agent binding a particular analyte isconjugated to an iCORE of a particular fragmentation signature, so thatthe analyte bound by the binding agent can be identified by thefragmentation signature of the iCORE.

Some aspects of this invention relate to biochemical encoding ofparameters, for example, of analytes, into mass tags, for example,iCOREs. In some embodiments, the biochemical encoding of an analyteparameter, e.g., the presence of an analyte in a sample or the amount ofan analyte in a sample, involves contacting the sample with a bindingagent (e.g., an antibody or antibody fragment) specifically binding theanalyte and bound to an iCORE the fragmentation signature is assigned tothe specific analyte bound by the binding agent, isolating or enrichingfor those binding agent molecules that have bound the analyte,optionally, releasing the iCORE from the isolated or enriched bindingagent molecules and subjecting the iCORE (released or not) to an MSassay. In other embodiments, the biochemical encoding is achieved bytranslating a parameter, for example, the presence of an analyte in asample, or the identity of a cell or tissue, into an iCORE signature bytagging the sample, cell, or tissue with a unique iCORE, for example, byspiking an iCORE into the sample, or by attaching (e.g., covalently viaa cleavable linker) an iCORE to a cell or tissue. In some embodiments,the biochemical encoding is carried out for multiple parameters inparallel, for example, by contacting a complex sample (e.g. a blood ortissue sample) containing or suspected to contain a plurality ofanalytes with a plurality of iCOREs bound to a plurality of bindingagents, wherein each binding agent specifically binds a particulartarget analyte and is conjugated to a specific iCORE with a uniquefragmentation signature. After isolation or enrichment of analyte-boundbinding agents and rescue of the conjugated iCOREs from the isolated orenriched binding agents, the presence or absence as well as the relativeor absolute amount of the target analytes can be determined by an MSassay, e.g., an LC MS/MS assay as described in more detail elsewhereherein.

A schematic of an exemplary biochemical encoding strategy in which thepresence of a plurality of analytes in a sample is translated intoiCOREs is described in FIG. 1 . In some embodiments, the translation ofa parameter into an iCORE comprises a step of deconvolution, forexample, a step of analyte enrichment from a complex biological sample.Deconvolution can increase assay sensitivity, specificity, and/oraccuracy. In some embodiments, a sample to be assessed is enriched foran analyte by contacting with an affinity agent that binds the analyte,specifically or non-specifically, and allows for enrichment of theanalyte, for example, by physical separation of the bound analyte fromunbound material. An affinity agent may be an agent thatnon-specifically binds the analyte based on a non-specific interaction,for example, a non-specific interaction based on the surface charge ofthe analyte. Such non-specific binding will typically result in a lowerlevel of deconvolution of the sample than the use of an affinity agentthat specifically binds the analyte, since non-specific binding isgenerally not restricted to the specific analyte, but also extends toother molecules of similar physiochemical properties. Alternatively, anaffinity agent may also be an agent that specifically binds the analyte.For example, an affinity agent may be a binding agent, for example, anantibody or antibody fragment, that is immobilized on a solid support,such as a bead or membrane surface. Solid supports useful for theseparation of materials bound to their surface from biological samplesare well known to those of skill in the art, and include, but are notlimited to, membranes, resins, beads, and the surface of plates, dishesand tubes.

In some embodiments, a complex sample is contacted for deconvolutionwith an affinity agent under conditions suitable for the affinity agentsto bind the analyte(s). Typically, the sample comprises a liquid phasethat is contacted with the affinity agent. In some embodiments, theaffinity agent, and any analyte(s) bounds to it are subsequently removedfrom the sample, for example, by physical separation, or aspiration ofliquid supernatant. In some embodiments, the affinity agent is washedafter separation to remove remaining residue of the biological sample onthe affinity agent for further deconvolution.

In some embodiments of biochemical encoding of analytes, a sample iscontacted with an affinity agent or a set of affinity agents thatbind(s) a set of analytes of interest, under conditions suitable for theaffinity agents to bind the respective analytes, and for the analytes tobecome immobilized on the affinity agent. In some embodiments, thesample is then de-convoluted, for example, by washing away any unboundmaterial. In some embodiments, the sample, deconvoluted or not, is thencontacted with a set of iCOREs conjugated to binding agents thatspecifically bind a set of analytes under conditions suitable for thebinding agents to bind their respective analytes. In some embodiments,those iCOREs conjugated to binding agents that have actually bound to ananalyte are subsequently isolated or enriched, for example, by removingany unbound iCOREs and binding agents from the sample. This “rescued”set of iCOREs represents a biochemical encoding of the analytes presentin the sample. In some embodiments, the iCOREs are conjugated to thebinding agents via a cleavable linker, and the process of biochemicalencoding of the analytes in the sample comprises a step of cleaving thelinker and releasing the iCOREs from the binding agents. In someembodiments, the rescued iCOREs are subjected to an MS assay, forexample, an MS/MS assay as described herein, to determine their identityand/or quantity, and thus the identity and/or quantity of the respectiveanalytes in the sample.

It will be appreciated, that translation for biochemical encoding is notrestricted to analytes, but can be applied to other parameters as well.For example, iCORE technology provides a possibility to track samples,cells, tissues, reagents, or molecules. As one example of biochemicalencoding for tracking purposes, iCORE technology can be used to encodesmall molecule compound identities, e.g., in the context of smallmolecule screens. Biochemical encoding of small molecule compoundidentities is particularly useful in the context of large combinatorialsmall molecule screens, where thousands, tens of thousands, or hundredsof thousands of small molecule combinations can be tracked without thelogistic problems commonly imposed by such screens. In some embodiments,each small molecule in such a screen is associated with an iCORE havinga unique fragmentation signature, for example, by simply adding theiCORE to the small molecule, thus generating a composition comprisingthe iCORE and the small molecule. In some embodiments, the compositioncomprises the iCORE in the small molecule at a specific, known ratio. Insome embodiments, the composition is then used in a combinatorialchemical screening assay, for example, in an assay in which multiplecombinations of multiple small molecule compounds in multiple dilutionratios are tested for a desired effect. The desired effect may be anyeffect for which small molecule libraries are currently being screened,including, but not limited to, a biological effect, such as theinduction of cell death in a cancer cell line, the inhibition ofaberrant cell proliferation, the induction or repression of theexpression of a specific gene, or the induction of epigeneticreprogramming. Many other desired effects for which small moleculecompound libraries can be screened will be apparent to those skilled inthe art, and the invention is not limited in this respect.

Biochemical encoding of each small molecule with a unique iCORE allowsfor the identification and/or the quantification of each small moleculecompound in a mixture or dilution series of small molecule compounds bysubjecting the mixture or dilution to an MS assay. Any specific iCOREdetected in the MS assay can then be traced back to the particular smallmolecule compound it encodes. If the concentration of the small moleculecompound was also encoded, for example, by generating the originaliCORE/small molecule compound mixture used in the screen to comprise aparticular ratio of the two components, the intensity of signal detectedfor the specific iCORE can be used to determine the concentration of thesmall molecule compound in the mixture or dilution of interest.

For example, a simple combinatorial small molecule screening assay maycomprise a combinatorial dilution series screen of 10 small moleculecompounds (SMC 1-10) at 10 different dilutions (D 1-10), in which allpossible mixtures of any or all of the ten compounds at any possibledilution ratios of the compounds are to be tested. For example, tenpotentially synergistic cancer drug candidates might be tested this wayfor their efficiency to kill cancer cells, e.g., to investigate whichcombination of these candidates at which ratio effects the greatestsynergy or the greatest biological effect. Even such a simple screenposes a significant logistic challenge in view of the necessity to mapthe contents and drug concentrations of multiple thousands of samples.Biochemical encoding of the drug candidates with only 10 iCOREs (e.g.,iCORE G1-10, assigned to CD1-10, respectively) circumvents the problemsassociated with this logistical undertaking. For example, each drugcandidate could be spiked with one of the iCOREs at a specific, knownratio of drug candidates and iCORE at the beginning of the experiment.The generation of dilution series and drug candidate mixtures can thenbe carried out as usual, but without the necessity to keep track of allpipetting steps and the resulting sample contents. After screen readout,for example after the detection of a sample showing the desiredbiological effect, the supernatant of the sample can be obtained, andsubjected to an MS assay. The MS assay will not only identify thepresence or absence of each of the drug candidates, but can also be usedto determine the ratios of the drug candidates identifies to be presentin the sample based on the known iCORE/drug candidate ratios in theoriginal spiked mixture at the beginning of the experiment.

For example, if in the hypothetical screen described above, the MS assaywould detect the presence of iCOREs 1, 4, and 7 at a ratio of 1:10:2 ina sample of interest, the presence of drug candidates 1, 4, and 7 can beinferred. It can further be inferred that drug candidate 1 was presentat the lowest concentration (e.g., D10), drug candidate 4 at the highestconcentration (e.g., D1), and drug candidate 7 at the second lowestconcentration (e.g., D9) of the dilution series. If molar ratios ofiCORE and drug candidate were known in the original spiked sample at thebeginning of the experiment, a determination of the concentration ofeach drug candidate may be possible directly from the MS data, withoutthe need to track dilutions.

Aside from circumventing the necessity to track thousands of pipettingsteps, drug candidate tracking with iCORE biochemical encoding duringcombinatorial screening allows for an assessment of the actualconditions in the sample of interest, which, for example, avoidserroneous results caused by pipetting errors. The particular iCOREs usedfor biochemical encoding and drug screening should be chosen to notinterfere with the process being probed. Peptide iCOREs are suitable fora variety of biochemical encoding applications during drug screening,since the peptides are inert to most biochemical processes. It will beapparent to those of skill in the art, that, depending on the length andthe conditions encountered by the iCOREs during the drug screeningprocess, useful iCOREs will have to exhibit sufficient stability towithstand the conditions encountered. In some embodiments, peptideiCOREs with improved stability are generated by using non-naturallyoccurring amino acid residues and/or peptides comprisingnon-hydrolyzable bonds. Peptides exhibiting increased stability, forexample, peptides comprising non-hydrolyzable bonds, are well known tothose of skill in the art, and the skilled artisan will be able toreadily ascertain methods and reagents for the generation.

In some embodiments, a method for tracking a cell, cell population,tissue, or compound by biochemical encoding via iCOREs is provided. Insome such embodiments, the method comprises conjugating a unique iCOREto a cell, cell population, or tissue, for example, by contacting thecell, population of cells, or tissue with an iCORE under conditionssuitable for the iCORE to bind to the cell, population of cells, ortissue. In some embodiments, an iCORE covalently bound to a bindingagent is used to achieve this conjugation. For example, in someembodiments, the iCORE is bound to a binding agent, for example, anantibody or antibody fragment, that specifically binds a surface marker,for example, a surface protein or polysaccharide marker of the cell,population of cells, or tissue. In other embodiments, a cell, cellpopulation, tissue, or compound is contacted with an iCORE comprising orconjugated to a reactive chemical moiety under conditions suitable forthe reactive chemical moiety to form a covalent bond with a surfacemarker of the cell, cell population, tissue, or with the compound. Insome embodiments, the compound is a compound that is used in thegeneration of or can be embedded into a hydrogel, for example, a PEGhydrogel as described in FIG. 7 . In some embodiments, the hydrogel isused in the generation of beads or in the production of a scaffold fortissue engineering, e.g., as a substrate for growing artificial,engineered tissues, for example, engineered organs, such asmicro-livers. The result of this approach is the production ofiCORE-tagged beads or scaffolds and, if such scaffolds are used for thegeneration of engineered organs, iCORE-tagged organs. In someembodiments, a plurality of different iCORE-tagged beads or engineeredorgans is subjected to different experimental conditions, e.g., theexposure to different growth factors and/or small molecule compounds, orgrowth factor and/or small molecule compound combinations at differentdilutions, and those beads or engineered organs exhibiting a desiredphenotype or parameter at the end of the experiment is identified byrescuing the iCORE tag from the desired bead or organ and subjecting itto an MS assay, e.g., an LC MS/MS assay.

In some embodiments, a set or library of iCOREs is provided thatcomprise or is conjugated to a reactive chemical group that can form acovalent bond to a peptide. Such reactive chemical groups are known tothose of skill in the art and include, but are not limited to thosereactive chemical groups conjugated or comprised in mass tags used iniTRAQ assays. In some embodiments, methods for the use of suchpeptide-binding iCOREs are provided, for example for the simultaneousanalysis of proteins in samples of different origin, for example, bloodor tissue samples from different subjects. In some embodiments, proteinsamples obtained from different subjects, for example, from differentexperimental mice, or from different tumor biopsies, are conjugated to aunique iCORE each. In some embodiments, a plurality of suchiCORE-labeled samples is then pooled and simultaneously analyzed in anMS assay, for example, an LC MS/MS assay. In some embodiments, the dataobtained from such simultaneous proteomics experiments is used tocompare protein expression levels across a large number of samples, forexample, across at least 10, at least 15, at least, 20, at least 25, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 150, at least 200, at least 250,at least 500, at least 750, at least 1000, or more than 1000 samples.The large number of possible unique fragmentation signatures that can beobtained from a single iCORE base sequence, for example, from a glu-fibamino acid sequence, allows for the unique labeling and subsequentsimultaneous analysis of such sample numbers, which is far beyond themultiplexing capabilities of the currently available technologies, e.g.,iTRAQ.

Some aspects of this invention provide multiplexed diagnostic assaysusing iCORE technology. Currently, many molecular biomarkers are knownthat are correlated with, and, thus, indicative of a disease orcondition in a subject. For example, a plethora of molecular biomarkershave been reported for diseases such as cancer, infectious disease,cardiovascular disease, renal disease, autoimmune disease, toxic states,and for the detection of certain side effects of drugs. Currentdiagnostic assays typically focus on one or only a few biomarkers todetect a specific disease or condition. For example, some cancers, suchas prostate and ovarian cancer are monitored by use of single biomarkersin the blood of subjects diagnosed or suspected to have such a cancer.Such diagnostic techniques are achieved, for instance using fluorescencedetection of molecular markers which are activated in a particulardisease state. Other diagnostic techniques to monitor molecules includethe use of gene arrays for stratifying breast cancer patients via uniquesignatures (Sotiriou C., Piccart, M. J., Taking gene-expressionprofiling to the clinic: when will molecular signatures become relevantto patient care?, Nat. Rev. Cancer, 7, 534-553, 2007) and sequencingtarget gene mutations to uncover the molecular lesions that predictglioblastoma response to EGFR kinase inhibitors (Mellinghoff, I. et al.,Molecular Determinants of the response of glioblastomas to EGFR KinaseInhibitors, NEJM, 353, 16, 2005). Both of the foregoing references areincorporated herein in their entirety by reference for their disclosureof diagnostic biomarkers.

Some of the reagents and methods for multiplex analyte detection andquantification in complex samples, for example, in a body fluid ortissue sample obtained from a subject, allow for the sensitive andaccurate assessment of multiple biomarkers simultaneously, thusproviding an avenue for the generation of clinically relevantmulti-parameter biomarker profiles of a single disease or condition orof a plurality of diseases or conditions from such a sample.

In some embodiments, iCORE technology as described herein is used toassess a plurality of biomarkers, e.g., the presence or absence or thequantity of an analyte in a biological sample obtained from a subject,such as a body fluid (e.g. blood, serum, urine, cerebrospinal fluid, orlymph) or tissue (e.g., tumor, malignant, neoplastic, hyperplastic,liver, kidney, lung, muscle, lymph node, or brain tissue). In someembodiments, the plurality of biomarkers comprises a panel of biomarkersknown to be associated with a particular disease or condition. In someembodiments, a diagnostic iCORE method is provided. In some embodiments,the method comprises obtaining a sample from a subject. In someembodiments, the subject is a human subject. In some embodiments, thesubject is a non-human mammal. In some embodiments, the method comprisesa deconvolution step in which target analytes are isolated or enriched,for example, by a deconvolution method as described elsewhere herein. Insome embodiments, the method comprises contacting the sample, whetherdeconvoluted or not, with a set or library of iCOREs conjugated to a setof binding agents specifically binding to the target analytes, asdescribed herein, under conditions suitable for the iCORE-conjugatedbinding agents to bind their respective target ligands. In someembodiments, the method comprises a step of isolating or enriching thebound iCORE-conjugated binding agents, for example, by washing offunbound binding agents. In some embodiments, the method comprisesrescuing the iCOREs conjugated to the analyte-binding binding agents,for example, by cleavage of a linker connecting the iCOREs to thebinding agents, as described elsewhere herein. In some embodiments, themethod comprises subjecting the rescued iCOREs to an MS/MS assay, forexample, an MS/MS assay as described herein. In some embodiments, themethod further comprises determining the signal obtained from eachspecific iCORE in the sample based on the results of the MS/MS assay. Insome embodiments, the method comprises determining the presence orabsence of the analyte represented or identified by the specific iCOREbased on whether or not a signal was obtained in the MS/MS assay thatidentified the specific iCORE. In some embodiments, the method comprisesquantifying the amount or concentration of the analyte in the sample, orrelative to the amount or concentration of other analytes in the sample,or relative to the amount or concentration of the same analyte in adifferent sample, for example, a reference or control sample, byquantifying the signal obtained from the specific iCORE in the MS/MSassay.

In some embodiments, the assessment of multiple biomarkers in a singlediagnostic assay increases the specificity, sensitivity, and/or accuracyof the assessment of the biomarkers and/or the diagnosis inferred fromthe assay. In some embodiments, the iCORE MS/MS assay also comprises theassessment of a control marker, for example, of an analyte or a set ofanalytes that are known to be virtually constant across healthy anddiseased states or across individuals. There are many protein biomarkerspresent in blood that are useful for assessment by iCORE MS/MS analyses.For example, in certain embodiments for ovarian cancer, 5-20 protein orpeptide biomarkers may be monitored (see, e.g., Petricoin, E., et. Al.,Use of proteomic patterns in serum to identify ovarian cancer, Lancet,359, 572, 2002, incorporated in its entirety by reference for disclosureof ovarian cancer biomarkers). Serum cytokines, of which IFN-gamma andTNF-alpha are normally classified, including, but not limited to, IL-1,Il-2, Il-4, Il-5, Il-6, Il-10, Il-12, Il-13, Il-17, Il-21, Il-23, MCP-1,TGF-beta, TNF-beta, TWEAK, GM-CSF, and Granzyme B, are useful formonitoring inflammatory processes involved in cancer, as well as hostresponses to vaccines, and infectious diseases. It will be apparent tothose of skill in the art that the biomarkers described herein are forillustration only and not meant to limit the invention in this respect.Any biomarker, and particularly, any peptide or protein biomarker can beassessed by iCORE MS/MS assays provided herein, and the invention is notlimited in this respect.

In some embodiments, iCORE technology is used in the context ofpersonalized medicine. For example, in some embodiments, a clinicalintervention, e.g., a drug, a treatment schedule, or a surgicalintervention, is chosen from a group of such interventions, based on theresults of an iCORE assay performed on a sample obtained from thesubject, for example, a blood or tissue sample assessed for a set ofbiomarkers, or based on analyte or enzymatic activity signaturesproduced from an in vivo iCORE assay, by methods described herein.

In some embodiments, iCORE technology is used to monitor a subject'sresponse to a treatment. For example, in some embodiments, iCORE assaysare performed repeatedly during the treatment of a diseased subject, forexample, in order to determine whether the assessed biomarkers areapproaching desired values, for example, values typically associatedwith a state of health. In some embodiments, a treatment regimen may beadjusted based on the results of an iCORE assay performed during thecourse of the treatment, for example, in order to increase the efficacyof the treatment or to decrease the dosage of a drug used to the minimaleffective dose in order to avoid undesired side effects.

In some embodiments, iCORE technology is used in clinical trials ofnovel drugs and drug candidates. Multiplex iCORE technology allows forthe assessment of a variety of disease-associated biomarkers, and, insome embodiments, also for the monitoring of a plurality of baseparameters, e.g. blood cholesterol, blood triacylglycerides, ketonebodies, and so forth, in a subject during a clinical trial. This allowsfor the comprehensive monitoring of multiple metabolic pathways during aclinical trial and the detection of side effects that may benon-symptomatic during the trail.

Mass spectrometry (MS) assays are well known to those of skill in theart. Of particular use in the context of some embodiments of iCOREtechnology and biochemical encoding described herein are tandem massspectrometry (MS/MS) assays.

Tandem mass spectrometry (MS/MS) is used to produce structuralinformation about a compound by fragmenting specific sample ions insidethe mass spectrometer and identifying the resulting fragment ions.Tandem mass spectrometry allows for specific iCOREs to be detected incomplex mixtures of iCOREs, for example, in complex sets or libraries ofiCOREs on account of their unique fragmentation signature, which resultsin a specific and characteristic fragmentation pattern.

MS/MS assays typically comprise two or more MS steps with some form offragmentation taking place between the steps. A tandem mass spectrometertypically comprises more than one analyzer, for example, two analyzers.In some embodiments, the analyzers are separated by a collision cellinto which an inert gas (e.g. argon, xenon) is admitted to collide withthe selected sample ions and bring about their fragmentation. However,some MS/MS assays can also be performed on certain single analyzer massspectrometers such as ion trap and time-of-flight instruments, forexample, by using a post-source decay experiment to effect thefragmentation of sample ions.

In some embodiments, because both the masses of the parent peptide aswell as the reporter ions of individual iCOREs are known, the detectionand quantification of the iCOREs in a complex sample can be achieved byselected reaction monitoring (SRM), also referred to as multiplereaction monitoring (MRM) (see, e.g., Lange, V., Picotti, P., Domon, B.,Aebersold, R., Selected reaction monitoring for quantitative proteomics:a tutorial, Molecular Systems Biology 4:222, 2008, the entire contentsof which are incorporated herein by reference). A typical SRM experimentis enabled by the unique capability of a triple quadrupole (QQQ) MS forquantitative analysis in which the first and third quadrupoles act asfilters to specifically select predefined m/z values corresponding tothe peptide ion and a specific fragment ion of the peptide.

For example, in some embodiments, for iCOREs based on 10-plex glu-fibiCORE library (G10), the first quadrupole serves to collect the parentGlu-fib peptide (e.g. 789.85 m/z doubly charged), the second quadrupolefragments, while the third quadrupole acts to filter an individualreporter ion (e.g. 683.3 m/z) (FIG. 14 ). In some embodiments, suchprecursor/fragment ion ‘transitions’ (e.g. 789.85 to 683.3 m/z) aremonitored over time, which results in high selectivity and sensitivity,as co-eluting background ions are filtered out effectively with thisapproach.

In some QQQ embodiments, unlike other MS techniques (e.g. Q-TOF), nofull mass spectra are recorded and the non-scanning nature of QQQanalysis translates into 1-2 orders of magnitude increase in sensitivityover full scan techniques. Because isotopic amino acids can beselectively incorporated by the user to generate unique fragment ionswith iCORE encoding, multiple unique precursor/fragment ion transitionscan be designed into a family of iCOREs. For example, in FIGS. 2 and 3 ,the transitions are defined from the precursor peptide to the y7 familyof fragment ions, while in FIG. 13 c,d,e and FIG. 14 , the transitionsare defined form the precursor peptide to the y6 family of fragmentions.

In some embodiments, the first analyzer is used to select user-specifiedsample ions arising from a particular iCORE. These chosen iCORE ions,for example, the ions of the glu-fib iCOREs described herein, pass intothe collision cell, are bombarded by the gas molecules which causefragment ions to be formed, and these fragment ions are then separatedaccording to their mass to charge ratios, by the second analyzer. Thefragment ions arise directly from the parent iCORE ions, and thusproduce a fingerprint pattern specific to the compound underinvestigation. (see FIG. 3 I. for data obtained in the first round of MSfrom a library of glu-fib iCOREs; and FIG. 3 II. for data obtained inthe second round (after fragmentation) from the same library).

In some embodiments, the first analyzer allows the transmission of alliCORE ions, whilst the second analyzer is set to monitor specific iCOREfragment ions, for example, the glu-fib iCORE y₇ ions, as described inFIGS. 2 and 3 , which are generated by bombardment of the sample ionswith the collision gas in the collision cell. This strategy isparticularly useful in embodiments, where a set of isobaric iCOREs isused, which fragment to produce distinct, but similar fragment ions.Using this strategy allows for monitoring only the part of the spectrumspanning the particular masses of the iCORE ions, e.g. the spectrum fromabout 812 to about 822 Da in the example using iCOREs G1-G10 asdescribed in FIG. 3 . This, in turn allows for a drastic reduction inscanning time, because the rest of the spectrum can be ignored.

In some embodiments, both analyzers are static and only specific parentions are transmitted through the first analyzer and only specificfragments arising from these parent ions are measured by the secondanalyzer. The parent ion properties of any iCORE provided herein can becalculated according to methods well known to those of skill in the art.Additionally, any iCORE can be tested for its specific ionization andfragmentation signature in an MS/MS test run. Since the structure andMS/MS behavior of each iCORE is either known or can readily becalculated or established, this type of MS/MS assay can be employed todetermine the presence or absence of a specific iCORE ion in a matrix orfragment ions, for example, a matrix of fragment ions originating from acomplex isobaric iCORE library in a multiplex iCORE assay as provided byaspects of this invention.

In some embodiments, peptide iCOREs, sets or libraries of peptideiCOREs, and methods for the use of peptide iCOREs are provided. It isknown to those of skill in the art that peptides fragment in areasonably well-documented manner in MS/MS assays (see, e.g., P.Roepstorrf, J. Fohlmann, Biomed. Mass Spectrom., 1984, 11, 601; and R.S. Johnson, K. Biemann, Biomed. Environ. Mass Spectrom., 1989, 18, 945;the entire contents of which are incorporated herein by reference). Insome embodiments, the peptides fragment along the peptide backbone (see,e.g., A. E. Ashcroft, P. J. Derrick in “Mass Spectrometry of Peptides”ed. D. M. Desiderio, CRC Press, Florida, 1990; the entire contents ofwhich are incorporated herein by reference). There are three differenttypes of bonds that can fragment along the amino acid backbone: theNH—CH, CH—CO, and CO—NH bonds. Each bond breakage gives rise to twospecies, one neutral and the other one charged, and only the chargedspecies is monitored by the mass spectrometer. The charge can stay oneither of the two fragments depending on the chemistry and relativeproton affinity of the two species. Hence there are six possiblefragment ions for each amino acid residue and these are labeled as a, b,and c ions, and x, y, and z ions, with the a, b, and c ions having thecharge retained on the N-terminal fragment, and the x, y, and z ionshaving the charge retained on the C-terminal fragment. The most commonfragmentation occurs at the CO—NH bonds of the peptide backbone, whichgive rise to the b and/or the y ions. FIG. 2 shows an exemplarynomenclature of 13 y-ions originating from a parental glu-fib iCOREsequence. Since the mass difference of adjacent fragment ions correlatesto the mass of the respective amino acid residue missing in the shorterion, e.g., the difference in the mass of the y₉ and y₁₀ ions is the massof the D residue present in the y₁₀ but not the y₉ ion, the peaksrepresenting each y ion can be identified. In the exemplary MS/MSexperiment shown in FIG. 2 , y ions y₃-y₁₁ could unambiguously beidentified.

An example of an MS/MS spectrum obtained from a set of 10 isobariciCOREs is illustrated in FIG. 3 . The first round of MS generated onesingle peak, consistent with the isobaric character of the iCOREs (FIG.3 I.). The iCORE ions were fragmented and the size of the fragmentsanalyzed by the second analyzer to produce the MS/MS spectrum depictedin FIG. 3 . II.). The iCOREs fragmented predominantly at the CO—NH bondsions y ions y₃-y₁₁ could unambiguously be identified. A close-up of theregion spanning the mass of the possible y₇ ions shows that all teniCOREs were detected with highly similar signal strength, indicatingthat all fragment ions were present at about the same concentration.This indicates that there is no fragmentation bias in iCOREs ofdifferent fragmentation signature, which allows iCORE technology to beused in sensitive analyte quantification MS/MS assays.

In some embodiments, the iCOREs are polynucleotides or polysaccharides.MS/MS-based assays oligonucleotide and oligosaccharide fragmentidentification are well known to those of skill in the art and aresimilar to those used in peptide MS/MS assays. For a general overview ofMS/MS assays useful for the detection and/or quantification of iCOREsprovided herein, for example, of peptide, oligonucleotide, and/oroligosaccharide iCOREs, see, e.g., S. Pomerantz, J. A. Kowalak, J. A.McClosky, J. Amer. Soc. Mass Spectrom., 1993, 4, 204; “An Introductionto Biological Mass Spectrometry”, C. Dass, Wiley, USA, 2002; “TheExpanding Role of Mass Spectrometry in Biotechnology”, G. Siuzdak, MCCPress, San Diego, 2004; “Ionization Methods in Organic MassSpectrometry”, A. E. Ashcroft, Analytical Monograph, Royal Society ofChemistry, U K, 1997; www.astbury.leeds.ac.uk (A. E. Ashcroft's MS webpages and tutorial); Chapter 9, pages 415-452 of “Mass Spectrometry: ATextbook” by Jurgen H. Gross, Springer; 2nd ed. edition (Mar. 1, 2011),ISBN-10: 9783642107092; U.S. Pat. Nos. 5,885,775; 7,412,332; 6,597,996;and “Mass Spectrometry: Clinical and Biomedical Applications Volume 1(Modern Analytical Chemistry)” by Dominic M. Desiderio, Springer; 1edition (Jan. 31, 1993), ISBN-13: 978-3642107092; the entire contents ofall of which are incorporated herein by reference).

Some aspects of this invention provides kits of reagents useful forcarrying out iCORE-based MS assays. Typically, a kit comprises acontainer housing an iCORE reagent, or a set or library of suchreagents, as described herein. In some embodiments, a kit also includesinstructions or labels describing the use of the iCORE reagents.

In some embodiments, iCORE reagents described herein, for example,iCOREs conjugated to a reactive chemical moiety, or iCOREs conjugated toa binding agent specifically binding a target ligand, or sets orlibraries of such iCORE reagents, are assembled into kits for clinicaland/or research applications. A kit may be designed to facilitate use ofthe methods described herein by clinicians and/or researches and cantake many forms. Each of the iCORE reagents comprised in the kit, whereapplicable, may be provided in liquid form (e.g., in solution), or insolid form, (e.g., a dry powder). A kit may have a variety of forms,such as a blister pouch, a shrink wrapped pouch, a vacuum sealablepouch, a sealable thermoformed tray, or a similar pouch or tray form,with the accessories loosely packed within the pouch, one or more tubes,containers, a box or a bag. In some embodiments, a kit comprising iCOREreagents as provided herein further comprises additional components thatare useful in performing the iCORE-based assays provided herein. Suchadditional components may include, but are not limited to deconvolutionreagents (e.g., binding agents specifically binding the set of analytestargeted by the iCORE reagents comprised in the kit, for example,immobilized to a solid support, such as beads (e.g., magnetic beads), amembrane, glass slide, resin, or plastic vessel surface), buffers (e.g.,wash buffers, binding buffers), and enzymes (e.g., proteases cutting acleavable linker connecting iCOREs to binding reagents).

EXAMPLES Example 1: Materials and Methods

Peptide and Ab peptide synthesis. All peptides were synthesized by Fmocchemistry in house by the biopolymers facility at the SwansonBiotechnology Center (MIT). Isotope-labeled amino acids were purchasedfrom Cambridge Isotopes. The photosensitive linker(3-Na-Fmoc-Amino-3-(2-nitrophenyl)propionic acid) was purchased fromAdvanced Chemtech. Detection antibodies against human IFN-gamma andTNF-alpha (eBioscience, clones 4S.B3 and Mab11) were first reacted withSIA (Pierce) at a 50:1 mole ratio. Following removal of excess SIA,peptides were incubated with Abs at a 20:1 mole ratio. Excess peptideswere removed by size filtration spin filters (30 k mwco, Amicon).

Ab-bead synthesis. Tosyl-activated magnetic beads (Myone, Invitrogen)were reacted with anti-human IFN-gamma and anti-human TNF-alpha(eBioscience, clones NIB42 and Mab1 respectively) according tomanufacturer's instructions. Briefly, 1 mg of either antibody wasbuffered exchange into borate buffer (0.1M Sodium borate, pH 9.0) priorto incubation with the beads in conjugation buffer (0.1M Borate, 1Mammonium sulphate, pH 9.0). After overnight incubation at 37° C.,remaining tosyl groups were saturated by incubating the beads in 1Mtris. The Ab-labeled beads were stored in PBS prior to experiments.

Serum assays. Recombinant human IFN-gamma (eBioscience) was spiked into10% bovine serum in PBS. This solution was then incubated withanti-IFN-gamma-beads for 1 hour at 37° C. The beads were then washedtwice with PBS before incubation with an equimolar solution ofiCORE-labeled IFN-g and TNF-a for another hour at 37° C. After excessantibodies were removed by washing with 0.1% BSA PBS, the bound antigensand antibodies were eluted in 5% acetic acid. The solution was thenexposed to UV light (365 nm) for 30 min. (CL-1000, UVP) to liberate thephotocaged iCORE codes into solution. The sample was then analyzed by LCMS/MS.

Nanomaterial synthesis. Nanoworms were synthesized according topreviously published protocols.²⁵ Peptides were synthesized at MIT(Swanson Biotechnology Center); isotopically labeled Fmoc amino acidswere purchased from Cambridge Isotopes and3-Nα-Fmoc-Amino-3-(2-nitrophenyl)propionic acid from Advanced Chemtech.Amine-terminated NWs were first reacted with Vivotag 680 (Perkin Elmer)to enable in vivo imaging, before reacted with SIA (Pierce) to introducesulfhydryl-reactive handles. Cysteine-peptides and PEG-SH were thenmixed with NWs overnight at room temperature (95:20:1 molar ratiorespectively) and excess peptides were removed by size filtration.Peptide-NW stock solutions were stored in PBS at 4° C.

In vitro protease assays. For substrate screening, Fl-peptide-NWs (2.5μM by peptide) were mixed with recombinant MMP-2/8/9 (R&D Systems),MMP-7/14 (AnaSpec, Inc.), Thrombin, Tissue Factor, Factor Xa, orCathepsin B (Haematologic Technologies, Inc.) in a 96-well plate at 37°C. in activity buffers according to manufacturer's instructions andmonitored with a microplate reader (SpectroMax Gemini EM). For MSanalysis, equimolar iCORE-encoded NWs (Table) were incubated withproteases for 2.5 hrs at 37° C. Cleavage fragments were purified fromNWs by size filtration before UV treatment (365 nm, CL-1000 UVcrosslinker, UVP). Reporters were then dried by speed vacuum centrifugeand stored at 4° C.

In vivo imaging. All animal work was approved by the committee on animalcare (MIT, protocol #0408-038-11). FVB/NJ mice (Jackson Labs) were fedwith 0.1% w/w DDC (Sigma) rodent chow for three weeks (Research Diets).Fibrotic and age control animals were i.v. infused with VivoTag-680labeled reagents and visualized by IVIS imaging (Xenogen). For tumorxenografts, LS 174T cancer cells lines were maintained in 10% FBS EMEMand inoculated s.c. (5×10⁶/flank) in NU/NU mice (Charles River) prior toimaging.

Characterization of models. For in situ zymography, fibrotic sectionswere covered with 90 μl solution of 0.5% w/v low melt agarose (Sigma) inMMP activation buffer (50 mM Tris, 150 NaCl, 5 mM CaCl2, 0.025% Brij 35,pH 7.5) with 10 μl of DQ-gelation (1 mg/ml, Invitrogen) and Hoechst dyeat 37° C. Slides were solidified at 4° C. and then incubated at roomtemperature overnight to promote gelation proteolysis by tissueproteases. To quantify hepatic collagen, tissue from the right and leftlobes (250-300 mg) were hydrolyzed in 5 ml of 6 N HCl at 110° C. for 16hours followed by hydroxyproline quantification as previouslydescribed.⁴³ To quantify CEA, blood was collected from tumor animalsinto Capiject microtubes (Terumo) to isolate serum before ELISA analysis(Calbiotech). For immunofluorescence analysis, equimolar NW cocktails (5μM/peptide) were administered in Fibrotic FVB/NJ or tumor bearing Nudemice. After perfusion, livers or tumors were fixed in 4% PFA, froze forsectioning, and stained for F4/80 (AbD Serotec), MMP-9 (R&D Biosystems),CD31 (Santa Cruz Biotechnologies) and/or FITC (Genetex) before analyzedby fluorescence microscopy (Nikon Eclipse Ti).

Collection of urinary peptides. Mice were intravenously infused with 200μl of PBS containing equimolar NW cocktails (5 μM per peptide) withEDTA-free protease inhibitor tablets (Roche) to isolate MMP activity.Mice were placed over 96-well plates surrounded by cylindrical sleevesfor urine collection. To prevent further reporter degradation, voidedsamples were spiked with EDTA+ complete protease inhibitors (Roche)immediately after collection. To quantify urinary fluorescence, 2 μL ofeach sample was incubated with magnetic beads (Dynal) coated with α-FITCantibodies (Genetex) in 50 μl binding buffer (100 nM NH₄OAc, 0.01%CHAPS) for 1 hr at 37° C., washed twice with 100 mM NH₄OAc, and elutedtwice with 15 μl of 5% acetic acid. Samples were neutralized with 2 MTris and quantified by microplate fluorimetry. For iCORE analysis,samples were irradiated with UV for 30 min. before TCA precipitation(20% final volume) to remove proteins. Soluble fractions were applied toC18 reverse phase columns (Nest Group) and eluted via step gradients of20% ACN increments in 0.1% formic acid. 60% ACN fractions containingGlu-fib peptides were collected and dried by vacuum centrifuge.

LC MS/MS analyses. Peptide samples were reconstituted in 5% ACN, 0.1%formic acid and analyzed at MIT or the Taplin MS facility (HarvardMedical School). At MIT, peptides were captured and eluted from a C18nanoflow HPLC column (75 μm internal diameter Magic C18 AQ, MichromBioResources, Inc.) at a flow rate of 300 nL/min usingwater-acetonitrile solvent system with 0.1% formic acid. ESI massspectrometry was carried out on a QSTAR Elite QTOF mass spectrometer (ABSciex). At Harvard, samples are reconstituted in 2.5% ACN, 0.1% formicacid. Samples are injected using a Famos autosampler (LC Packings) intoan Agilent 1100 HPLC prior to mass analysis on a LTQ-Orbitrap (ThermoElectron). To account for discrepancies in urine volumes andconcentration, peak intensities of individual reporters were scaledrelative to its respective total iCORE ion current before normalizationagainst control samples to account for technical and age-relatedvariations.

Statistical Analyses. Pearson's correlation coefficients betweendifferent protease profiles were calculated with MatLAB. ANOVA analyseswere calculated with GraphPad 5.0 (Prism). For ROC analysis, risk scorefunctions were first estimated by logistic regression on individualbiomarkers followed by ROC curve analyses of single or biomarkercombinations (SigmaPlot).

Example 2: Detection of TNF-alpha and IFN-gamma in a Liquid Sample

A liquid sample comprising of recombinant human IFN-gamma spiked into10% bovine serum to simulate a complex biological sample was made. Asshown in FIG. 1 a , the sample was first deconvoluted by contacting thesample with antibodies conjugated to the surface of beads. Specificbinding to anti-human-TNF-alpha and anti-human-IFN-gamma antibodiesbound to the surface of the beads resulted in immobilization of thetarget analyte IFN-gamma on the bead surface. The beads were thenseparated from the liquid sample by a magnet to remove the liquid phaseand any unbound materials comprised in any sample residue remaining onthe beads were washed away from the beads by washing the beads with awash buffer. The immobilized analytes bound to the binding agents on thebead surface were then contacted with another set of antibodies againstTNF-α and IFN-g that were conjugated to a set of iCOREs via aphotocleavable linker(dE-⁺³G-⁺⁶V-dN-dD-dN-dE-dE-G-F-F-dS-A-dR-(ANP)-K(FAM)-G-G-C (SEQ ID NO:37), dE-⁺²G-⁺⁶V-dN-dD-dN-dE-dE-G-F-F-dS-⁺¹A-dR-(ANP)-K(FAM)-G-G-C (SEQID NO: 37), respectively).

In this experiment, the set comprised two antibodies, one specificallybinding IFN-gamma and the other specifically binding TNF-alpha. Bothantibodies were conjugated to isobaric iCOREs, with the iCOREs bound tothe antibodies binding IFN-gamma having a different fragmentationsignature than the iCOREs bound to the antibodies binding TNF-alpha. Anyunbound antibodies were then removed from the beads by washing the beadswith wash buffer. The beads were then incubated in a stripping solutionto elute the bound material, and the resulting solution collectedfollowing removal of beads by a magnet. The photocleavable linkers werethen cleaved by exposure to UV light. The iCOREs rescued in this mannerwere then subjected to an LC MS/MS assay. FIG. 1 b shows the resultingpeaks of the second round MS (after fragmentation). The target analyteIFN-gamma was detected in the sample while endogenous levels ofTNF-alpha, or non-specific binding contributed to the first peak.Because the iCOREs were isobaric, the signal intensity of each iCORE candirectly be translated into a relative abundance of the target analyte.Accordingly, IFN-gamma was far more abundant than TNF-alpha in thesample analyzed, representing a robust signal to noise of approximately10.

Example 3: Utilization of a Canonical Peak from Glu-Fib MS/MS Spectra asa Reporter

Glu-fib was evaluated for its usefulness as a base sequence for thegeneration of an isobaric iCORE library. Glu-fib is a 13 amino acidpeptide of the sequence EGVNDNEEGFFSAR (SEQ ID NO: 1). The fragmentationspectrum resulting from collision induced disassociation results in thegeneration of y-type ions. FIG. 2 is a typical spectrum to illustratethe resulting y-type ions (y1-y13) and their relative intensities.

The propensity of glu-fib to fragment along certain peptide bonds wasexploited to construct 10 mass codes centered on the y₇ ‘reporter’ ion(EGFFSAR (SEQ ID NO: 6)) by enriching the truncated sequence with heavyamino acids to produce variants differentiated by 1 Da each. Thisintroduced mass shift is then compensated by isotope enrichment withinthe ‘balance’ fragment (EGVNDNE (SEQ ID NO: 7)). As a result, eachpeptide is characterized by an identical nominal mass and a distinct y₇fragment ion. Thus this iCORE set is isobaric, with a shared parent massof 1579.68 Da. The mass of the b₇ fragment (balance) was calculated foreach balance as M_(b7(i))=(M_(b7)+n)−i and the mass of each y₇-fragmentas M_(y7(i))=(M_(y7)+i), with M_(b7)=767.3 and M_(y7)=812.38. Thecalculated masses of four iCOREs of the set are given in the table onthe lower right of FIG. 2 . The set of isobaric iCOREs was thensubjected to an MS/MS assay. The data obtained is shown in the MS/MSgraph in FIG. 3 .

Example 4: MS/MS Data from Isobaric iCORE Libraries

A 10-plex library of glu-fib iCOREs was subjected to an MS/MS assay. Theresults are depicted in FIG. 3 . The upper panel of the figure providesthe sequences and fragmentation signatures of the iCOREs in the set(iCOREs G1, G2, G3, G4, G5, G6, G7, G8, G9, and G10). The upper panelalso shows the extracted ion chromatograph obtained from the 10-plexiCORE library (G1-G10) after the first round of MS. Only one peak wasobserved (m/z=789.7-790.0).

Accordingly, each iCORE is mass indistinguishable from the multiplexedset as well as undifferentiable by chromatography, which, in someembodiments, is beneficial for quantification, which is consistent withthe calculations in Example 3. The middle panel of FIG. 3 shows MS/MSresults following fragmentation of the peak observed in the upper panel.The results demonstrate that, also consistent with the observations ofExample 3, y-ions y₃-y₁₁ could be identified. Importantly, magnificationof the m/z window around the predicted m/z values of the y₇fragmentation ions generated revealed individual, distinguishable peakscorresponding to individual iCORE fragmentation signatures (lowerpanel). Relative quantification of the signals obtained for G1-G10revealed that the relative representation of the iCOREs in the originalsample (equal amounts of each iCORE) was closely mirrored in the peakpattern obtained, indicating that the method is not only fit forqualitative detection (absence/presence of an analyte), but also forquantitative and comparative quantitative studies.

Example 5: Quantitative iCORE MS/MS Assays

In order to investigate whether relative quantities of iCORE abundancesin a sample are accurately reflected in MS/MS assays such libraries aresubjected to, a sample comprising a 10-plex iCORE library (G1-G10), asdescribed in Example 4, was created, in which the different iCOREs werecomprised at different relative abundances in defined ratios. Theoriginal sample comprised the following iCORE stoichiometry:G1:G2:G3:G4:G5:G6:G7:G8:G9:G10 were at a ratio of1:2:3:4:5:10:10:5:3:2:1. The sample was subjected to an MS/MS analysisand the results are shown in FIG. 4 . The bar graph shows a goodcorrelation of the original stoichiometry and the measured abundances ofthe iCOREs. Further analysis of expected (theoretical) and observed(extracted) abundances of each iCORE demonstrate that the MS/MS resultsclosely resemble the actual abundances in the sample. The correlationcoefficient of the observed and the expected data set was calculated tobe R²=0.99. These results indicate that multiplex iCORE assays aresuitable for quantitative analysis and that iCORE abundance ismaintained and accurately reflected after multiplex MS/MS assays.

Example 6: Linear Dynamic Range of iCORE MS/MS Assays

In order to investigate the dynamic range in which iCOREs could bereliably detected and quantified, a dilution series of a 10-plex iCORElibrary, as described in Example 4, was generated. The library compriseeach iCORE (G1-G10) at the same abundance. Five diluted samples, with aconcentration of 10⁻⁷M (100 nM), 10⁻⁸M (10 nM), 10⁻⁹M (1 nM), 10⁻¹⁰M(100 pM), and 10⁻¹¹M (10 pM), respectively, were then subjected to anMS/MS assay. The observed intensities for each iCORE at eachconcentration were plotted in relation to the concentration of theiCOREs in the dilution sample. FIG. 5 shows that all iCOREs wereaccurately detected at all concentrations, indicating that sensitivityof iCORE MS/MS assays and the linear dynamic range, in which actualiCORE abundances in the sample are accurately reflected in the observedsignal intensity, extends to at least from the picomolar range to thenanomolar range, spanning at least five orders of magnitude. Theseresults indicate that accurate detection of iCOREs in a multiplex MS/MSassay is possible at high sensitivity, with even picomolar levels ofiCOREs being detectable at an accuracy allowing for quantitativeanalysis.

Example 7: Efficiency of iCORE Release from Covalently Bound SubstrateVia Photocleavage

In order to evaluate the efficiency of release of iCOREs covalentlybound to a substrate, for example, a binding agent specifically bindinga target analyte, a glu-fib-based iCORE was generated and conjugated toa substrate via a covalent bond. The iCORE was connected to thesubstrate, another peptide via a photocleavable linker, thus generatinga photo-caged, or photocleavable, iCORE-substrate tandem peptide. Thephoto-caged tandem peptide was exposed to UV light of ˜350 nm wavelength for a time (30 min) sufficient for photocleavage of the linker,which resulted in a release of the iCORE from the tandem peptide asshown in the upper panel of FIG. 6 . The process of photocleavage wasmonitored by LC MC (lower panel). The data obtained demonstrate that thephoto-caged tandem peptide (I, m/z=881.7) was quantitatively cleaved andclose to 100% of the theoretically recoverable amount of the releasediCORE (II, m/z=785.4) was rescued. These results indicate thatphotocleavage of iCOREs covalently bound to other molecules, e.g., otherpeptides, such as binding agents, or hydrogel-forming molecules, via aphotocleavable linker, can be efficiently released by exposure to UV,and can be quantitatively rescued and accurately quantified in LC MSassays.

Example 8: Labeling of Molecules with iCOREs by Conjugation to ReactiveMoieties

In order to investigate whether iCOREs could be used to covalently labelother molecules, glu-fib iCOREs are conjugated to a reactive chemicalgroup able to form a covalent bond to a compatible chemical moiety undercertain conditions. For this, a glu-fib iCORE is attached to a reactivechemical moiety able to form a covalent bond to a PEG monomer whenexposed to UV light in the presence of PEG monomers. As depicted in FIG.7 , UV exposure results in the formation of a glu-fib iCORE-labeled PEGpolymer, in this case a hydrogel, in which the PEG scaffold iscovalently bound by iCOREs.

This proof-of principle experiment demonstrates that substrates can becovalently labeled with iCOREs and that iCORE-labeled hydrogels, useful,for example, as scaffolds for tissue engineering, can readily beachieved.

Example 9: Mass-Encoded Synthetic Biomarkers for Multiplexed UrinaryMonitoring of Disease

A protease sensing platform comprising multiplexed synthetic biomarkersfor the detection of disease was developed. FIG. 8 shows a schematic ofthis approach. FIG. 8 a shows a synthetic biomarker library comprised ofmass encoded substrate peptide library conjugated onto nanowormnanoparticles. Administration of NW cocktails in whole animals leads toaccumulation in disease tissues (b). Local proteases cleave peptidefragments that subsequently filter into urine. Photo-caged massreporters are released upon exposure to UV-light and quantified byliquid chromatography tandem mass spectrometry (c).

To establish a protease sensing platform, peptide substrates wereidentified that would be susceptible to cleavage by extracellularproteases associated with liver fibrosis and cancer. Fluorescein-labeledderivatives of ˜50 candidate peptide sequences²⁰⁻²⁴ were synthesized andconjugated to PEG-coated, long-circulating iron oxide nanoworm²⁵ (NW)nanoparticles (FIG. 9 a,b,c) and incubated with recombinant proteasescommonly overexpressed in these diseases (e.g. Matrixmetalloproteases(MMPs), cathepsins) as well as proteases normally present in blood (FXa,Tissue factor (TF), thrombin) to evaluate substrate specificity andaccessibility of surface-conjugated peptides to proteases. FIG. 9 showslong-circulating iron oxide nanoworm chaperones. NW size distribution asdetermined by dynamic light scattering of dextran-coated, iron oxidenanoworms is shown in (a). Absorbance spectra of free NWs (red) and NWsconjugated with Fluorescein-labeled peptides (˜500 nm) and Alexa 647(blue) are shown in (b). In vivo clearance kinetics of PEG-coated (blue)and PEG-free (red) NWs conjugated with substrate peptides are shown in(c). Pegylated NWs extended circulation half-life by ˜4 hours.

Using this assay, increases in sample fluorescence were observed uponproteolytic cleavage of the tethered fluorescent peptides resulting fromabrogation of homoquenching between adjacent fluorophores. Initialreaction velocities were extracted for each protease-substratecombination and compiled for comparative analysis (FIG. 10 a,b ). FIG.10 illustrates selecting protease-sensitive substrates for NW-chaperonedurinary trafficking. Kinetics of peptide-NW cleavage monitored byfluorimetry are shown in (a). Specific protease-substrate combinationsled to rapid activation. FIG. 10 b shows a heat map comparison ofinitial cleavage velocities for different substrate-proteasecombinations grouped according to activity and specificity. IVIS in vivoimaging of DDC treated and control animals following intravenousinjection of VivoTag-680 labeled Glu-fib peptides, (d) peptide-free NWs,or (e) peptide-conjugated NWs.

From this set, 10 peptides with robust protease susceptibility wereselected to serve as a peptide-NW library (Table 1).

TABLE 1iCORE-encoded peptide biomarker library. Individual probe substrates encodedwith photo-caged isobaric mass codes for quantification by LC MS/MS.Synthetic Biomarker Library

Substrate Isobaric mass code

y

 reporter [y

+H

] G1 e

G

VndneeGFfsAr-X-K(FAM) PQGIWGQ e

G

VndneeGFfsAr GPSAE 683.4 G2 e

G

Vndnee

GFfsAr-X-K(FAM) LVPRGSG e

G

Vndnee

GFfsAr

GFIsA 884.4 G3 e

G

Vndnee

GFfsAr-X-K(FAM) PVGLIG e

G

Vndnee

GFfsAr

GFsAr 685.4 G4 eG

Vndnee

GFfs

Ar-X-K(FAM) PWGIWGQG eG

Vndnee

GFfs

Ar

GFfs

Ar 686.4 G5 eG

VndneeGFfs

Ar-X-K(FAM) PVPLSLVM eG

VndneeGFfs

Ar GFfs

Ar 687.4 G6 e

G

Vndnee

GFfs

Ar-X-K(FAM) PLGLRSW e

G

Vndnee

GFfs

Ar

GFfs

Ar 688 4 G7 e

GVndneeG

FfsAr-X-K(FAM) PLGVRGK e

GVndneeG

FfsAr G

FfsAr 889.4 G8 e

GVndneeG

Ffs

Ar-X-K(FAM) f(Pip)RSGGG e

GVndneeG

Ffs

Ar G

Ffs

Ar 690.4 G9 e

GVndnee

G

FfsAr-X-K(FAM) fPRSGGG e

GVndnee

G

FfsAr

G

FfsAr 691.4 G10 eGVndnee

G

FfsAr-X-K(FAM) f(Pip)KSGGG eGVndnee

G

FfsAr

G

FfsAr 692.4

indicates data missing or illegible when filed

Sequences of G1-G10 correspond, from top to bottom, to SEQ ID NO: 8-SEQID NO: 17, respectively. Substrate sequences correspond, from top tobottom, to SEQ ID NO: 18-SEQ ID NO: 27, respectively. The isobaric masscodes correspond to SEQ ID NO: 28, and the y6 reporters correspond toSEQ ID NO: 29.

In order to design a system that would probe diseased microenvironments,the biodistribution of each system component (i.e. peptide andnanoparticle) was investigated in disease models in vivo. Here, a mousemodel of liver fibrosis was selected in which FVB/NJ mice fed with3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) develop progressiveliver disease as a result of chronic bile duct injury²⁶ (FIG. 11 a,b,c),leading to fibrotic and proteolytically active livers (FIG. 12 b,c,d).

FIG. 11 illustrates urinary biomarkers of hepatic fibrosis andresolution in DDC-treated mice. FIG. 11 a shows the induction of liverfibrosis and NW administration timeline. FIG. 11 b shows quantificationof total liver collagen by hydroxyproline analysis. DDC treatment led to˜3 fold increase in liver collagen by week 3 (***P<0.001) and a ˜30%drop between week 7 and 11 (*P<0.05) that remained above pre-treatmentvalues (*P<0.05) (One-way ANOVA, Tukey post test, n=3, s.e.m.). Siriusred histochemistry of liver sections (scale bar=50 μm) indicated thepresence of fibrotic extensions emanating from portal triads at week 3,persisting to week 7, and resolving by 11 weeks (c). IVIS in vivoimaging showed urinary accumulation of ensemble reporter library inDDC-treated animals (d). Kinetics of urinary accumulation quantified byα-FITC immunoprecipitation (3 wk timepoint, *P<0.05, Two-way ANOVA,Tukey post test; error bars=s.e.m.) are shown in (e). FIG. 11 f showsbox and whisker plots of individual iCORE peak intensities plotted asDDC over control at 0, 3, 7, and 11 weeks (*P<0.05, **P<0.01; repeatedmeasures ANOVA, Tukey post test; n=10). FIG. 11 g shows ROC curves ofsingle, double and triple combinations of fibrosing biomarkers withassociated area under curves (AUC). (h) ROC curves of single, double andtriple combinations of resolving biomarkers with associated area undercurves (AUC).

FIG. 12 illustrates immunofluorescence of liver sections. FIG. 12 ashows periportal images of macrophage (red) and NW (green) infiltrateinto fibrosing zones. Arrows highlight areas of colocalization. FIG. 12b shows periportal images of MMP9 (red) and NW (green). MMP9 expressionwas not found in healthy livers (c). FIG. 12 d shows Fluorescence imageof periportal zone after gelatin in situ zymography. Scale bars=100 μm.

To evaluate peptide trafficking, the peptide glutamatefibrinopeptide B(Glu-fib, EGVNDNEEGFFSAR, SEQ ID NO: 1) was selected as a prototypicurinary marker because its endogenous derivative (fibrinopeptide B) isbiologically inert and normally filters freely into urine when releasedduring coagulation.²⁷ Fluorophore-labeled Glu-fib administeredintravenously (i.v.) efficiently filtered into urine in both fibrosingand healthy animals with undetectable liver homing (FIG. 10 c ). Incontrast, larger peptide-free NWs (˜40 nm hydrodynamic radius, FIG. 9 a) homed to the liver (FIG. 10 d ), consistent with the size-dependenttrafficking of nanomaterials and a renal clearance threshold of ˜5 nmfor inorganic nanoparticles.²⁸ Importantly, in animals treated withfluorophore-labeled peptides conjugated to NWs, significant NW-mediatedpeptide homing was observed to both fibrosing and healthy livers leadingto a strong increase in urinary fluorescence in diseased animalsresulting from renal filtration of cleaved peptide fragments (FIG. 10 e).

Profiling Protease Activities by Mass Spectrometry

Despite the multiplexing advantages of mass-encoding, one challenge ofdetecting protease activity by MS is that peptide substrates in complexproteolytic environments can be cleaved at multiple sites by promiscuousproteases and truncated by exoproteases^(4, 29) to produce diverse poolsof poorly defined fragments that confound mass analysis. Here,well-defined mass reporters to encode a substrate library were designed.In light of the favorable renal clearance properties of Glu-fib,d-isomer rich derivatives of Glu-fib were appended to the N-termini ofeach protease substrate to serve as protease resistant mass reportersand to promote renal filtration upon substrate cleavage and release fromNWs (Table 1). These tandem peptides were further modified with internalphoto-labile residues³⁰ to enable the recovery of well-defined Glufibpeptides by photolysis from complex urinary cleavage fragments followingin vivo proteolysis. To test this construct, a model photo-caged tandempeptide was synthesized (compound I, FIG. 13 a ). Consistent withpreviously published reports on nitrophenyl groups, exposure of compoundI (triply charged, 881.7 m/z; FIG. 13 b , top panel) to UV lighttriggered peptide cleavage, resulting in the appearance of doublycharged, acetamide-terminated Glu-fib (785.4 m/z; FIG. 13 b , bottompanel).

FIG. 13 shows photo-caged iCORE libraries for multiplexed profiling ofprotease activities by LC MS/MS. FIG. 13 a illustrates the structure oftandem peptide (compound I) containing an internal UV-sensitive linker.Shown here is the structure of free Glu-fib (compound II) generatedafter photolysis (˜350 nm). FIG. 13 b shows LC MS spectra of compound Ibefore (top, triply-charged m/z: 881.7) and after (bottom,doubly-charged m/z: 785.4) exposure to UV light. FIG. 13 c shows a10-plex isobaric peptide library derived from Glu-fib. FIG. 13 d showsan extracted ion chromatogram of an equimolar 10-plex iCORE mixture(789.80-789.90 m/z). The entire multiplexed set was chromatographicallyindistinguishable. FIG. 13 e shows an iCORE MS/MS spectrum followingcollision induced disassociation. Individual reporters were identifiedvia unique y6 reporter ions (683.3-692.3 m/z) each differentiable by asingle mass unit. FIG. 13 f shows an iCORE MS/MS spectrum followingincubation of a 10-plex, iCORE-encoded peptide-NW cocktail withrecombinant MMP9.

FIG. 14 illustrates Isobaric COded REporter (iCORE) mass encoding. FIG.14 a shows an MS/MS spectrum following collision induced disassociationof Glu-fib. Peaks correspond to c-terminal, y-type peptide fragments.FIG. 14 b lists 10 isotopic analogs and corresponding masses producedvia isobaric encoding. Each sequence was constructed by selectivelyenriching the balance or reporter regions with ‘heavy’ amino acids toproduce unique y6 reporter ions while maintaining a uniform total mass.

In order to design an extensible encoding strategy for the library ofprotease substrates, the ability of isobaric mass encoding^(31, 32) wasinvestigated to be extended to peptide scaffolds such as the urinaryreporter Glu-fib to produce a family of mass reporters. Thedistinguishing feature of an isobaric encoding strategy is thatindividual members of a family of reporters share a parent mass tofacilitate efficient peptide collection by MS, but can be subsequentlyidentified via unique MS/MS ions upon fragmentation. It was firstdetermined that Glu-fib fragments into C-terminal y-type ions (FIG. 10 a) and 10 mass codes were constructed centered on the y6 ion (GFFSAR, SEQID NO: 4) by enriching the hexamer with heavy amino acids to producevariants differentiated by 1 Da each (FIG. 10 b ). This introduced massshift was then balanced by isotope enrichment within the remainder ofthe peptide (EGVNDNEE, SEQ ID NO: 5). As a result, each peptide wascharacterized by an identical nominal mass and a distinct y6 fragmention. This encoding method was termed “isobar COded REporters” (iCORE).To validate this approach, an equimolar 10-plex iCORE mixture (FIG. 13 c) was analyzed by LC MS/MS and the entire peptide library was found toinitially appear as a single, unresolved peak (extracted ionchromatogram, 789.95±0.5 m/z, FIG. 13 d ) but following fragmentation,to resolve into a 10 peak spectrum with no fragmentation bias(683.4-692.4 m/z, FIG. 15 , FIG. 13 e ). To remove confounding peakoverlap arising from naturally occurring isotopes (e.g. 13C), iCOREpeptides were selectively fragmented via a unit mass window centered onthe precursor ion (FIG. 16 a ) to minimize the signal from naturallyoccurring isotopes to ˜5% of the parent peak (FIG. 16 b ). Thus, insamples spiked with reporters at defined ratios (1:2:3:5:10:10:5:3:2:1),a linear correlation was observed between peak intensity andstoichiometry in both unmodified and peak-subtracted analysis (n=3,R2=0.99 and R2=0.99 respectively, FIG. 17 a,b,c). FIG. 17 demonstratesthat iCORE LC MS/MS analysis is quantitative. FIG. 17 a shows an MS/MSspectrum of a 10-plex mixture of iCORE reporters combined in a1:2:3:5:10:10:5:3:2:1 ratio. Extracted peak intensities were highlycorrelated with the input reporter stoichiometry (r2=0.99; n=3; errorbars=s.e.m.) (b). To account for naturally occurring isotopes (FIG. 16), individual reporter intensities were modified by subtracting 5% ofthe prior reporter intensity (c). Compensated reporter ratios alsostrongly correlated with reporter ratios (r2=0.99; n=3; errorbars=s.e.m.). All subsequent samples were peak-adjusted to reflectcontributions from naturally occurring isotopes.

To evaluate the ability of iCORE multiplexing to simultaneously reporton the activities of many protease-substrate combinations, a 10-plex,equimolar cocktail of iCORE encoded peptide-NWs was treated withrecombinant MMP9 (Table 1). Following incubation, cleavage products wereisolated by size filtration and exposed to UV-light prior to MS/MSanalysis.

Notably, collective substrate activities were translated into distinctiCORE landscapes characterized by markedly different y6 reporterintensities (FIG. 13F). This library was applied to several otherrecombinant proteases (FIG. 18 a ) and no strong correlation was foundbetween different iCORE protease profiles (i.e. MMP2, MMP9, MMP12, andthrombin) as determined from Pearson's correlation analysis (FIG. 18 b), illustrating the ability of iCORE-encoded NWs to monitor manyprotease and protease-substrate combinations uniquely.

Example 10: Monitoring Hepatic Fibrogenesis and Resolution

Liver fibrosis is a wound healing response to chronic liver injury andresults in the buildup of scar tissue that can lead to cirrhosis, liverfailure and cancer.¹⁸ The dynamics of extracellular matrix accumulationsuch as collagen is largely driven by fibrogenic hepatic stellate cellsand myfibroblasts, and matrix remodeling proteases such as MMPs andtheir inhibitors. The current gold standard for monitoring is a needlebiopsy followed by histological analysis; however, this technique isinvasive, confounded by high sampling heterogeneity, carries a finiterisk of complications and cannot be performed frequently as needed (e.g.monitoring antifibrotic therapies).³³ Noninvasive assays includingultrasound imaging, elastography, and serum biomarkers are limited bytheir low accuracies and limited prognostic utility.³⁴ Thus, thereremains an urgent need for noninvasive biomarkers to replacebiopsy-based monitoring, identify and validate new antifibrotic agents,and to support clinical decision making.³⁵ Here, it was sought toidentify synthetic biomarkers with the capacity to monitor liverfibrosis, and to extend the DDC-induced model to include both fibrosingand resolving disease (FIG. 11 a,b,c).

The in vivo trafficking and ability of iCORE-encoded syntheticbiomarkers to produce an ensemble urinary signal during fibroticprogression was determined. Intravenous administration of afluorophore-labeled, 10-plex peptide-NW cocktail resulted in a 2-foldincrease in urinary fluorescence in animals treated with DDC for 3 weeks(*P<0.05, Twoway ANOVA; FIG. 11 d,e ). Immunofluorescence imaging ofliver sections revealed that most NWs escaped sequestration by residentmacrophages (FIG. 12 a , arrow), infiltrating freely into the parenchymaand into actively fibrosing periportal zones characterized by punctateMMP9 expression (FIG. 12 b,c ). Similar patterns appeared in sectionstreated with DQ-gelatin substrates (FIG. 12 d ) which permit in situvisualization of active collagen-degrading proteases.

Collectively, these results indicated that NWs home to fibrosing livermicroenvironments containing active proteases. In order to determine thebehavior of individual reporters to identify sensitive and specificsynthetic biomarkers, the processes of fibrosis and resolution wereprobed by iCORE mass analysis. Mice treated transiently with DDC for 3weeks followed by restoration of DDC-free chow develop distinctfibrosing and resolving windows (0-3 and 7-11 weeks respectively, FIG.11 a ) as verified macroscopically by Sirius red collagen staining ofliver sections (FIG. 11 c ), and by hydroxyproline analysis whichquantifies total tissue collagen (FIG. 11 b ). With this treatmentregime, liver collagen increased˜3-fold compared to pretreatment levelsafter 3 weeks on DDC, persisted from week 3-7 after initial removal ofDDC, and significantly decreased from week 7-11 after sustained DDCwithdrawal (*P<0.05, ***P<0.005, n=3).

Thus, to monitor the transitions between fibrosing and resolvingdisease, NWs were administered at 0, 3, 7, and 11 weeks into DDC-treatedand age-matched control animals followed by iCORE MS/MS analysis. Theresulting activities of individual biomarkers displayed markedlydivergent kinetics (FIG. 11 f ). Biomarkers G3 and G4 both stronglyincreased relative to pretreatment baselines, reaching a plateau by week11 despite staggered onset at week 7 and 3 respectively. G5 and G6showed opposing kinetics, significantly decreasing at week 3 beforeeither gradually returning to pretreatment intensities (G5) orpersisting to week 11 (G6). Interestingly, G7 tracked with the kineticsof DDC treatment, elevating sharply at week 3 followed by a rapidreversal at week 7. All remaining biomarkers (G1, G2, G8, G9 and G10)did not deviate from initial pretreatment activities (G1-G10; *P<0.05,**P<0.01, repeated measures ANOVA, Tukey post test, n=10). Importantly,all biomarkers in control animals also did not significantly depart frombaseline (FIG. 19 ).

The diagnostic performance of these biomarkers was determined byperforming receiver operating characteristic (ROC) analyses forindividual as well as biomarker combinations. ROC curves characterizethe sensitivity and specificity of a biomarker as a function of thediscrimination threshold by returning the area under the curve (AUC) asa performance metric with a baseline AUC of 0.5 representing a randombiomarker classifier (dashed line, FIG. 11 g,h ). Within the fibrosingwindow of 0-3 weeks, G5 displayed the highest AUC amongst the 10biomarkers (0.96, FIG. 20 ), which was further improved by including G7in a double biomarker combination (0.98), or G6 and G7 in a triplecombination (1.00), resulting in a perfect synthetic biomarkerclassifier for fibrosing disease in this model (FIG. 11 g ). Within theresolving time frame of 7-11 weeks, G1 led with AUC=0.73 (FIG. 21 ),which was significantly improved via the dual combination of G1+G9(AUC=0.9) and finally the triple combination G1+G7+G9 (AUC=0.91) (FIG.11 h ).

Collectively, these experiments demonstrate that liver fibrosis andresolution are revealed by distinct collections of synthetic biomarkers,and that multiplexed combinations allow the highest diagnosticperformance—illustrating the ability of this platform to noninvasivelyilluminate otherwise inaccessible aspects of liver disease evolution.

Example 11: Early Detection of Colorectal Tumors

When diagnosed prior to systemic dissemination, many primary tumors canbe effectively treated with conventional clinical interventions. ³⁶However, most clinically-utilized biomarkers lack the diagnosticaccuracy and sensitivity to discriminate small tumors, and it remainsunclear whether current endogenous blood biomarker strategies targetingshed or processed byproducts of cancerous tissue can be sufficientlyimproved for early detection.¹⁹ Moreover, it is becoming apparent thatindividual serum biomarkers (e.g. CA-125 for ovarian, PSA for prostatecancer) do not possess the necessary sensitivity and specificityrequired for early detection, and that panels of biomarkers are mostlikely required.³⁷

Here, it was investigated whether panels of synthetic urinary biomarkerscould be readily adapted to allow earlier and more accurate detection ofcancer compared with single clinical blood biomarkers, e.g., becausenanoparticles can be passively targeted to tumors to sample proteasesthrough fenestrated angiogenic tumor vessels.¹⁶. In order to explorethis concept, athymic nude mice were used bearing LS174T xenografttumors, a human colorectal cancer (CRC) cell line³⁸ that secretes theblood biomarker carcinoembryonic antigen (CEA) as a model system (FIG.22 ). FIG. 23 a shows a timeline of LS 174T colorectal cancer cellinoculation and NW administration in Nude mice. FIG. 23 b illustratesmacroscopic quantification of tumor growth (n=5, s.d.), and FIG. 23 cshows circulating levels of CEA in tumor and control animals analyzedevery third day post tumor implantation by ELISA (**P<0.01, Two-wayANOVA, Tukey post test). IVIS in vivo imaging showed urinaryaccumulation of ensemble reporter library in tumor-bearing animals (d).FIG. 23 e displays quantification of urinary fluorescence byFITC-immunoprecipitation (*P<0.05, Two-way ANOVA, Tukey post test; n=5,s.d.). FIG. 23 f shows ROC curves of a single, double and triplebiomarker combination with associated AUC (n=16; 8 ctrl, 8 tumor), andFIG. 23 g shows ROC comparison between triple biomarker combination(G1+G2+G3) with serum CEA at day 10. Following implantation, tumorengraftment was monitored by sampling serum every 3 days and analyzingfor CEA by ELISA (FIG. 23 b,c ). At day 10, CEA levels wereinsufficiently elevated to distinguish tumor from control animals,corresponding to an average tumor burden of ˜130 mm³ (spherical diameterd ˜6.3 mm). Tumors permitted to grow further were readily detected byCEA analysis (day 13 and 16), representing a limit of detection of ˜330mm3 (d ˜8.6 mm) (**P<0.01, Two way ANOVA, n=5). In order to determinewhether synthetic urinary biomarkers could outperform serum CEA, thefluorophore-labeled, 10-plex peptide-NW library described herein wasadministered at day 0 and 10. Ex vivo imaging of excised tumors andimmunofluorescence analysis of corresponding sections indicated that NWsreadily homed to the extravascular milieu following i.v. administration(FIG. 24 ). Despite the inability of CEA to indicate the presence ofcancer at day 10, ensemble peptide cleavage resulted in a strong 2-foldelevation in urine fluorescence (FIG. 23 d,e ), allowing the detectionof tumors ˜2.5-times smaller than CEA (130 vs. 330 mm³) by urinaryfluorescence alone. To determine the full diagnostic potential ofmultiplexed analysis, individual biomarkers were quantified by iCOREMS/MS. Similar to the liver studies described herein, the classifyingpower of the highest performing single biomarker (G1, AUC=0.78, FIG. 25) steadily improved in double (G1+G2, AUC=0.88) and triple biomarkercombinations (G1+G2+G3, AUC=0.89) (n=16, FIG. 230 . This triplecombination significantly outperformed serum CEA, which detected diseasepoorly with an AUC of 0.61 at day 10 (FIG. 23 g ).

Discussion

Despite the fact that many new features of disease are rapidly beingdiscovered with global profiling approaches, the development of thisknowledge into next-generation biomarkers remains fundamentally limitedby many technical and biological challenges intrinsic to endogenoustargets. Ideally, a candidate biomarker would be secreted at high levelsrelative to native background, remain stable or persistent incirculation until detection, be readily accessible from compositionallysimple host fluids, and be able to discriminate disease with highsensitivity and specificity. In practice, such parameters are difficultto improve or control, and many promising biomarkers fail duringrigorous evaluation for clinical translation.

Here, a system of synthetic biomarkers was devised with the capacity to(i) amplify biomarker levels through substrate turnover by targetingaberrant protease activities, (ii) release stable, d-isomer enrichedmass reporters designed to be detected within a narrow mass window freeof host molecules, (iii) trigger reporter clearance from blood intourine to reduce matrix complexity and to facilitate facile extraction,and (iv) simultaneously profile libraries of candidate syntheticbiomarkers in vivo to identify multiplexed combinations for highlysensitive and specific diagnosis. The work described herein shows thatby engineering exogenous agents to interrogate diseased tissues, keybiological and transport challenges can be separately addressed prior tointegration, resulting in synthetic biomarkers that can be rapidlydesigned, tested and identified for distinct diseases.

The liver studies described herein demonstrate the potential of thistechnology for monitoring both liver fibrosis and resolution. Currently,the needle biopsy remains the gold standard despite the fact that biopsyresults are often variable because only a small part of the liver issampled and can lead to inaccurate diagnosis or repeat biopsies.Furthermore, gross architectural changes frequently occur at a longertime scale compared with alterations in protease activities as occursduring resolution, making it challenging to predict patient trajectorybased on histological analysis. Notably, this study showed thatnanoscale agents penetrate throughout the liver, delineated a panel offibrosing and resolving biomarkers (0-3 and 7-11 weeks respectively),and even revealed biomarkers between week 3 and 7 (e.g. G3, G7) thatcould potentially represent an anticipatory signature in advance ofresolution since liver sections within this time window wereindistinguishable by clinically-utilized histological or matrixquantification assays. Early predictive biomarkers of fibroticresolution would accelerate the rate in which new anti-fibrotic drugsare identified in preclinical and clinical studies by quicklydifferentiating potential responders from non-responders with identicalhistology.

Currently, little clinical data exists correlating the size of tumors tocirculating biomarker levels and it remains unclear whether small tumors(<1 cm³) can be reliably detected. Recently, Gambhir and colleagues¹⁹have estimated via mathematical modeling that solid tumors couldpotentially remain undetectable for 10-12 years and reach a sphericaldiameter >2.5 cm before blood biomarkers could indicate disease. Inorder to be useful for early detection, current approaches dependent onendogenous species will need to be greatly improved. In comparison, thisstudy showed that multiplexed synthetic biomarker panels can detect CRCtumors earlier and more precisely than serum CEA, enabling the detectionof tumor burdens ˜2.5-fold smaller. It further illustrated thatrelatively small diseased sites (˜130 mm³) outside of the liver areaccessible for interrogation, opening the possibility of detectingdisease at sites currently challenging to probe with near-infraredimaging (e.g. deep seeded tumors) or with other imaging modalities (e.g.diseased tissues with poor MRI or CT contrast such as livermetastases).³⁹

The technology described herein can be readily extended to encompassadditional diseases and alternative applications. Given the cumulativewealth of approved and experimental nanoparticle formulations, the workdescribed herein is transferrable to other nanomaterials and scaffoldsthat can actively target or transport peptide cargo to different organs,types of vasculature, and tissue depths.^(40, 41) Further, the iCOREmass-encoding scheme disclosed herein can be extended to create hundredsof orthogonal codes by incorporating additional isotope-enriched aminoacids and by making use of distinct parent peptides. A larger encodinglibrary would not only enable the simultaneous monitoring of hundreds ofsynthetic biomarkers, but could additionally allow the identities ofdysregulated proteases to be revealed by mathematical deconvolution⁴² toovercome the challenges of designing specific substrates for promiscuousproteases. A collection of diverse delivery strategies, broadermultiplexing capabilities, and precise mathematical algorithms wouldprovide rich opportunities for systems-level monitoring of disease andclearly elucidate the roles that complex protease networks play in heathand disease.

REFERENCES

-   [1] Sawyers, C. L. The cancer biomarker problem. Nature 452, 548-552    (2008).-   [2] Hanash, S. M., Pitteri, S. J., and Faca, V. M. Mining the plasma    proteome for cancer biomarkers. Nature 452, 571-579 (2008).-   [3] Sreekumar, A. et al. Metabolomic profiles delineate potential    role for sarcosine in prostate cancer progression. Nature 457,    910-914 (2009).-   [4] Villanueva, J. et al. Differential exoprotease activities confer    tumor-specific serum peptidome patterns. J. Clin. Invest. 116,    271-284 (2006).-   [5] Surinova, S. et al. On the development of plasma protein    biomarkers. J. Proteome Res. 10, 5-16 (2011).-   [6] Schwarzenbach, H., Hoon, D. S. B., and Pantel, K. Cell-free    nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11,    426-437 (2011).-   [7] Moon, P.-G., You, S., Lee, J.-E., Hwang, D., and Baek, M.-C.    Urinary exosomes and proteomics. Mass Spectrom. Rev. 30, 1185-1202    (2011).-   [8] Nagrath, S. et al. Isolation of rare circulating tumour cells in    cancer patients by microchip technology. Nature 450, 1235-1239    (2007).-   [9] Lutz, A. M., Willmann, J. K., Cochran, F. V., Ray, P., and    Gambhir, S. S. Cancer screening: a mathematical model relating    secreted blood biomarker levels to tumor sizes. PloS Med. 5, e170    (2008).-   [10] Anderson, N. L. and Anderson, N. G. The human plasma proteome:    history, character, and diagnostic prospects. Mol. Cell. Proteomics    1, 845-867 (2002).-   [11] Haun, J. B. et al. Micro-nmr for rapid molecular analysis of    human tumor samples. Sci. Transl. Med. 3, 71ra16 (2011).-   [12] Fonovic, M. and Bogyo, M. Activity-based probes as a tool for    functional proteomic analysis of proteases. Expert Rev. Proteomics    5, 721-730 (2008).-   [13] Baruch, A., Jeffery, D. A., and Bogyo, M. Enzyme activity—it's    all about image. Trends Cell. Biol. 14, 29-35 (2004).-   [14] Hilderbrand, S. A. and Weissleder, R. Near-infrared    fluorescence: application to in vivo molecular imaging. Curr. Opin.    Chem. Biol. 14, 71-79 (2010).-   [15] Braet, F. and Wisse, E. Structural and functional aspects of    liver sinusoidal endothelial cell fenestrae: a review. Comp.    Hepatol. 1, 1 (2002).-   [16] Jain, R. K. and Stylianopoulos, T. Delivering nanomedicine to    solid tumors. Nat. Rev. Clin. Oncol. 7, 653-664 (2010).-   [17] López-Otín, C. and Bond, J. S. Proteases: multifunctional    enzymes in life and disease. J. Biol. Chem. 283, 30433-30437 (2008).-   [18] Schuppan, D. and Afdhal, N. H. Liver cirrhosis. Lancet 371,    838-851 (2008).-   [19] Hori, S. S. and Gambhir, S. S. Mathematical model identifies    blood biomarker-based early cancer detection strategies and    limitations. Sci. Transl. Med. 3, 109ra116 (2011).-   [20] Bremer, C., Tung, C. H., and Weissleder, R. In vivo molecular    target assessment of matrix metalloproteinase inhibition. Nat. Med.    7, 743-748 (2001).-   [21] Kridel, S. J. et al. A unique substrate binding mode    discriminates membrane type-1 matrix metalloproteinase from other    matrix metalloproteinases. J. Biol. Chem. 277, 23788-23793 (2002).-   [22] Lutolf, M. P. et al. Repair of bone defects using synthetic    mimetics of collagenous extracellular matrices. Nat. Biotechnol. 21,    513-518 (2003).-   [23] Mahmood, U. and Weissleder, R. Near-infrared optical imaging of    proteases in cancer. Mol. Cancer Ther. 2, 489-496 (2003).-   [24] Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C.    Determination of protease cleavage site motifs using mixture-based    oriented peptide libraries. Nat. Biotechnol. 19, 661-667-   [25] Park, J.-H. et al. Systematic surface engineering of magnetic    nanoworms for in vivo tumor targeting. Small 5, 694-700 (2009).-   [26] Fickert, P. et al. A new xenobiotic-induced mouse model of    sclerosing cholangitis and biliary fibrosis. Am. J. Pathol. 171,    525-536 (2007).-   [27] Morris, T. A. et al. Urine and plasma levels of fibrinopeptide    b in patients with deep vein thrombosis and pulmonary embolism.    Thromb. Res. 110, 159-165 (2003).-   [28] Choi, H. S. et al. Renal clearance of quantum dots. Nat.    Biotechnol. 25, 1165-1170 (2007).-   [29] Villanueva, J., Nazarian, A., Lawlor, K., Yi, S. S.,    Robbins, R. J., and Tempst, P. A sequence-specific exopeptidase    activity test (sseat) for “functional” biomarker discovery. Mol.    Cell. Proteomics 7, 509-518 (2008).-   [30] Brown, B. B., Wagner, D. S., and Geysen, H. M. A single-bead    decode strategy using electrospray ionization mass spectrometry and    a new photolabile linker: 3-amino-3-(2-nitrophenyl)propionic acid.    Mol. Divers. 1, 4-12 (1995).-   [31] Ross, P. L. et al. Multiplexed protein quantitation in    Saccharomyces cerevisiae using amine-reactive isobaric tagging    reagents. Mol. Cell. Proteomics 3, 1154-1169 (2004).-   [32] Thompson, A. et al. Tandem mass tags: a novel quantification    strategy for comparative analysis of complex protein mixtures by    MS/MS. Anal. Chem. 75, 1895-1904 (2003).-   [33] Rockey, D. C., Caldwell, S. H., Goodman, Z. D., Nelson, R. C.,    Smith, A. D., and for the Study of Liver Diseases, A. A. Liver    biopsy. Hepatology 49, 1017-1044 (2009).-   [34] Popov, Y. and Schuppan, D. Targeting liver fibrosis: strategies    for development and validation of antifibrotic therapies. Hepatology    50, 1294-1306 (2009).-   [35] Bedossa, P., Dargère, D., and Paradis, V. Sampling variability    of liver fibrosis in chronic hepatitis C. Hepatology 38, 1449-1457    (2003).-   [36] Etzioni, R. et al. The case for early detection. Nat. Rev.    Cancer 3, 243-252 (2003).-   [37] Kulasingam, V., Pavlou, M. P., and Diamandis, E. P. Integrating    high-throughput technologies in the quest for effective biomarkers    for ovarian cancer. Nat. Rev. Cancer 10, 371-378 (2010).-   [38] D'Souza, A. L. et al. A strategy for blood biomarker    amplification and localization using ultrasound. Proc. Natl. Acad.    Sci. 106, 17152-17157 (2009).-   [39] Schima, W., Kulinna, C., Langenberger, H., and Ba-Ssalamah, A.    Liver metastases of colorectal cancer: US, CT or MR? Cancer Imaging    5 Spec No A, S149-S156 (2005).-   [40] Ruoslahti, E., Bhatia, S. N., and Sailor, M. J. Targeting of    drugs and nanoparticles to tumors. J. Cell Biol. 188, 759-768    (2010).-   [41] Sugahara, K. N. et al. Science 328, 1031-1035 (2010).-   [42] Miller, M. A. et al. Proteolytic activity matrix analysis    (prama) for simultaneous determination of multiple protease    activities. Integr. Biol. 3, 422-438 (2011).-   [43] Popov, Y., Patsenker, E., Fickert, P., Trauner, M., and    Schuppan, D. Mdr2 (abcb4)−/− mice spontaneously develop severe    biliary fibrosis via massive dysregulation of pro- and    antifibrogenic genes. J. Hepatol. 43, 1045-1054 (2005).

The entire contents of all references described or listed herein,including references [1]-[43] listed above, any references described inthe specification above, and any reference described below, areincorporated herein by reference, as if each and every individualreference was incorporated herein by reference. In case of a conflictbetween the teachings of any reference incorporated herein and thespecification, the specification shall control.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not limited in scope by the examples provided, since theexamples are intended as illustrations of various aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include the plural reference unless the context clearlyindicates otherwise. Thus, for example, a reference to “a cell” includesa plurality of such cells, and a reference to “an antibody” is areference to one or more antibodies and equivalents thereof known tothose skilled in the art, and so forth.

Where ranges are given, endpoints are included. For each range anembodiment in which the described parameter assumes a single valuewithin the range or a sub-range within the range is included.

Not all embodiments have been described and combination of a feature orof multiple features of any embodiment(s) described herein with anyfeature or multiple features of any other embodiment(s) described hereinis contemplated. Similarly, any limitations in any of the appendedclaims may be combined with any limitation in any of the other claims,or a combination of such limitations. Such combined embodiments have notbeen explicitly set forth to save space, but are expressly contemplatedto be within the scope of the present invention. Similarly, anyfeature(s) of any embodiment described herein may be excluded from thescope of the appended claims.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1-103. (canceled)
 104. A composition comprising a plurality ofisotope-labeled molecules wherein each isotope-labeled molecule of saidplurality of isotope-labeled molecules comprises: a) a carrier, and b) amass tag conjugated to said carrier via a cleavable linker, wherein saidcleavable linker is cleaved by a disease-associated protease.
 105. Thecomposition of claim 104, wherein said mass tag comprises an isobaricmass tag.
 106. The composition of claim 104, wherein said mass tagcomprises a peptide mass tag.
 107. The composition of claim 104, whereinsaid isotope-labeled molecules are configured for use in massspectrometry (MS).
 108. The composition of claim 107, wherein saidisotope-labeled molecules are configured for use inliquid-chromatography mass spectrometry (LC-MS).
 109. The composition ofclaim 107, wherein said isotope-labeled molecules are configured for usein tandem mass spectrometry (MS/MS).
 110. The composition of claim 104,wherein said disease-associated protease is selected from the groupconsisting of MMP2, MMP7, MMP9, MMP12, MMP14, tissue factor, factor Xa,cathepsin, and thrombin.
 111. The composition of claim 104, wherein eachisotope-labeled molecules of said plurality of isotope-labeled moleculesis the same.
 112. The composition of claim 104, wherein said pluralityof isotope-labeled molecules comprises at least 6 different masstag-conjugated cleavable linkers.
 113. The composition of claim 104,wherein said plurality of isotope-labeled molecules comprises at least10 different mass tag-conjugated cleavable linkers.
 114. The compositionof claim 104, wherein said plurality of isotope-labeled moleculescomprises at least 20 different mass tag-conjugated cleavable linkers.115. The composition of claim 104, wherein said cleavage occurs in vivo.116. The composition of claim 104, wherein said cleavage occurs invitro.
 117. The composition of claim 104, wherein said cleavage occursex vivo.
 118. The composition of claim 104, wherein said carriercomprises a particle.
 119. The composition of claim 118, wherein saidparticle comprises a microparticle or a nanoparticle.
 120. Thecomposition of claim 104, wherein said carrier further comprises abinding agent.
 121. The composition of claim 120, wherein said bindingagent is selected from a group consisting of an antibody, an antibodyfragment, an aptamer, and an adnectin.
 122. The composition of claim104, wherein said cleavable linker comprises a peptide.
 123. Thecomposition of claim 104, wherein each isotope-labeled molecule of saidplurality of isotope-labeled molecules further comprises a reactivechemical moiety.